Sequential changes in classes of RNA during compensatory growth of the kidney.

The synthesis of nuclear precursors of rRNA and of cytoplasmic mRNA has been estimated in mouse kidneys following contralateral nephrectomy. 1. The production of nuclear rRNA parallels mitotic activity in the proximal tubule cells. Its peak is 2 days after nephrectomy; there may be a second peak at 8 days. 2. The production of mRNA is almost the reciprocal of rRNA production. It is least at 2 days and most at 4 days after nephrectomy. It may also decrease immediately after nephrectomy. 3. Growing kidney cells may build a store of ribosomes before they elaborate mRNA. The sequential relation between rRNA and mRNA synthesis raises the possibility that production of rRNA could regulate the production of mRNA and that the stimulus to compensatory hypertrophy might need to act only long enough to set into motion the machinery for making rRNA, either directly or as a consequence of exhausting mRNA.

Ultrastructural studies of human lymphoid cells. mu and J chain expression as a function of B cell differentiation.

J chain expression was examined as a function of the stage in differentiation along the B cell axis in humans. Intracellular distribution of J and mu chains in leukemic HLA-DR+ null and pre-B cells, and in normal B cells stimulated with pokeweed mitogen (PWM) was determined by immunoelectron microscopy and radioimmunoassay (RIA). J chain was detected in leukemic null and pre-B cells on free and membrane-bound ribosomes in the cytoplasm, or on perinuclear cisternae. Mu chain was found on free ribosomes and ribosomal clusters in leukemic pre-B cells but was absent in the leukemic null cells. In pre-B cell lines, mu chain was seen within rough endoplasmic reticulum (RER) and the Golgi apparatus whereas J chain was not detected in these organelles. However, both mu and J chain were detected in RER and the Golgi apparatus of immature and mature plasma cells induced by PWM stimulation of normal peripheral blood lymphocytes. Low levels of J chain were also detected by RIA in lysates of leukemic null and pre-B cells. Most of the intracellular J chain became detectable after reduction and alkylation of cell lysates, and free J chain was not found in the culture supernatants. The amount of intracellular and secreted immunoglobulin-bound J chain increased dramatically after PWM stimulation of peripheral blood lymphocytes. The majority of J chain-positive cells seen over an 8 d culture interval were lymphocytes and lymphoblasts, while mu chain was found primarily in plasma cells. These results suggest that J chain expression precedes mu chain synthesis during B cell differentiation and that a combination of the two chains for secretion is not initiated until the onset of plasma cells maturation.

Occurrence, isolation, and characterization of polyribosomes in yeast.

This report details the procedural requirements for preparing cell-free extracts of yeast rich in polyribosomes. This enabled us to demonstrate the occurrence of polyribosomes in yeast, to show their role in protein synthesis, and to devise methods for their resolution and isolation. When certain precautions are met (the use of log phase cells, rapidly halting cell growth, gentle methods of disruption, sedimentation through exponential density gradients, etc.), individual polyribosome size classes ranging up to the heptosome can be fractionated and separated from their nearest neighbors. Larger size classes are resolved partially among themselves, free of smaller polyribosomes. This was confirmed by extensive electron micrographic studies of material from the various fractions obtained upon density gradient centrifugation of yeast extracts. Modifications of the gradients and procedure should allow fractionation and isolation of the larger polyribosomes, including those containing polycistronic messages. Yeast polyribosomes are disaggregated to single ribosomes by longer term grinding, cell disruption by the French pressure cell, the Hughes press, or by incubation with dilute RNAse. Yeast polyribosomes are active in the incorporation of amino acids into polypeptide; the single ribosomes exhibit only slight activity. The latter activity is probably due to the presence of a small fraction of monosomes still containing mRNA. Poly-U stimulates amino acid incorporation only in the single ribosomes.

Fine structure of protein-storing plastids in bean root tips.

The fine structure of leucoplasts in root tip cells of Phaseolus vulgaris L. has been studied in material fixed in glutaraldehyde followed by osmium tetroxide and poststained in uranyl acetate and lead citrate. Plastid development has been followed from the young stages in and near the meristematic region, through an ameboid stage, to the larger forms with more abundant storage products in the outermost cells. The plastids contain a dense stroma penetrated by tubules and cisternae arising from the inner membrane of the plastid envelope. Also located in the stroma are lamellae, ribosome-like particles, phytoferritin granules, and fine fibrils in less dense regions. In some elongate plastids microfilaments run lengthwise in the stroma near the surface. The same plastids store both starch and protein, but in a strikingly different manner. The starch is deposited in the stroma, while the protein always is accumulated within membrane-bounded sacs. These sacs arise as outgrowths from a complex of interconnected tubules which in turn appears to originate by coalescence and proliferation of tubules and cisternae arising from the inner plastid membrane. This "tubular complex" bears a strong resemblance to the prolamellar body of etiolated chloroplasts, but is smaller and ordinarily less regularly organized, and is apparently light-insensitive. Crystallization of the protein commonly occurs in the sacs and occasionally takes place within the tubules of the complex as well. The fine structure of the leucoplasts is discussed in relation to that of etiolated chloroplasts. Suggestions are made concerning the function of the tubular complex, role of the ameboid plastid forms, and manner of accumulation of the storage protein in the plastids.

Distribution of newly synthesized amylase in microsomal subfractions of guinea pigs pancreas.

Amylase distribution was studied in guinea pig pancreas microsomes fractionated by centrifuging, for 2 hr at 57,000 g in a linear 10 to 30% sucrose gradient, a resuspended high speed pellet obtained after treating microsomes with 0.04% deoxycholate (DOC).(1) Amylase appeared in the following positions in the gradient: (a) a light region which contained approximately 35% of total enzymic activity and which coincided with a monomeric ribosome peak; (b) a heavy region which contained approximately 10% of enzymic activity in a sharp peak but which had very little accompanying OD(260) absorption; (c) a pellet at the bottom of the centrifuge tube which contained approximately 20% of the enzymic activity. After 5 to 20 min' in vivo labeling with leucine-1-C(14), radioactive amylase was solubilized from these three fractions by a combined DOC-spermine treatment and purified by precipitation with glycogen, according to Loyter and Schramm. In all cases, the amylase found in the pellet had five to ten times the specific activity (CPM/enzymic activity) of the amylase found in the light or heavy regions of the gradient. The specific radioactivity (CPM/mg protein) of the proteins or peptides not extracted by DOC-spermine was similar for all three fractions. Hypotonic treatment of the fractions solubilized approximately 80% of the total amylase in the fraction from the heavy region of the gradient, but only approximately 20% of the amylase in the monomer or pellet fraction. Electron microscope observation indicates that the monomer region of the gradient contained only ribosomes, that the heavy region of the gradient contained small vesicles with relatively few attached ribosomes, and that the pellet was composed mostly of intact or ruptured microsomes with ribosomes still attached to their membranes. It is concluded from the above, and from other evidence, that most of the amylase activity in the monomer region is due to old, adsorbed enzyme; in the heavy region mostly to enzyme already inside microsomal vesicles; and in the pellet to a mixture of newly synthesized and old amylase still attached to ribosomes. Furthermore, the ribosomes with nascent, finished protein still bound to them are more firmly attached to the membranes than are ribosomes devoid of nascent protein.

In vitro crystallization of ribosomes from chick embryos.

A new two-dimensional ribosome crystal, having the tetragonal space group P42(1)2 (a = 593 A), has been grown from ribosome tetramers extracted from hypothermic chick embryos. It is of particular interest because of its larger size (up to 3 x 3 micron2) and greater stability compared to other related polymorphic forms, and because it can easily be grown in large amounts. X-ray diffraction shows the order in the crystal to extend to a resolution of at least 60 A. The crystalline ribosomes appear to contain a full complement of small and large ribosomal subunit proteins and an additional four proteins not characteristic of chick embryo polysomes.

Ribosome precursor particles in nucleoli.

Ribonucleoprotein (RNP) particles containing the precursors of ribosomal RNA were extracted from L cell nucleoli and analyzed under conditions comparable to those used in the characterization of cytoplasmic ribosomes. Using nucleoli from cells suitably labeled with (3)H-uridine, we detected three basic RNP components, sedimenting at approximately 62S, 78S, and 110S in sucrose gradients containing magnesium. A fourth particle, sedimenting at about 95S, appears to be a dimer of the 62S and 78S components. When centrifuged in gradients containing EDTA, the 62S, 78S, and 110S particles sediment at about 55S, 65S, and 80S, respectively. RNA was extracted from RNP particles which were prepared by two cycles of zonal centrifugation. The 62S particles yielded 32S RNA and a detectable amount of 28S RNA, the 78S structures, 32S RNA and possibly some 36S RNA, and the 110S particles, a mixture of 45S, 36S, and 32S RNA's. When cells were pulsed briefly and further incubated in the presence of actinomycin D, there was a gradual shift of radioactivity from heavier to lighter particles. This observation is consistent with the scheme of maturation: 110S --> 78S --> 62S. The principal buoyant densities in cesium chloride of the 110S, 78S, and 62S particles are 1.465, 1.490, and 1.545, respectively. These densities are all significantly lower than 1.570, which is characteristic of the mature large subunit of cytoplasmic ribosomes, suggesting that the precursor particles have a relatively higher ratio of protein to RNA, and that ribosome maturation involves, in addition to decrease in the size of the RNA molecules, a progressive decrease in the proportion of associated protein.

Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma. II. General characterization of organelle nucleic acids.

Polytoma obtusum has a main band DNA (alpha) with a buoyant density in CsC1 of rho = 1.711 g/ml and a light DNA satellite (beta) with rho = 1.682 g/ml. beta-DNA was substantially enriched in a fraction containing small leucoplast fragments and some mitochondria, which was obtained in a pellet sedimenting between 3,000 g and 5,000 g. A crude mitochondrial pellet was also obtained by sedimenting at 12,000 g to recover particulates remaining in the supernate after 10 min at 5,000 g. This fraction contained a third DNA component (gamma) with rho = 1.714 g/ml. We have concluded that the leucoplasts of P. obtusum contain the beta-DNA (1.6882) and the mitochondria possess the gamma-component (1.714). Two distinct classess of ribosomes were isolated and separated by sucrose density gradients, a major 79S species and a minor species at 75S. The major species possessed the 25S and 18S ribosomal RNA (rRNA), characteristic of cytoplasmic ribosomes, and these particles co-sedimented in sucrose gradients with the 79S cytoplasmic ribosomes of Chlamydomonas reinhardtii. The minor species was present in about 2% of the total ribosomal population but showed an eight-to-ninefold enrichment in the leucoplast pellet, suggesting that it was of organelle origin. These 73S particles had RNA components migrating very closely with the 18S and 25S species of the 79S ribosomes, but the base composition of the rRNA from these two classes of ribosomes was significantly different; the rRNA from the 79S ribosomes had a G+C mole ratio of 50.0%, while the rRNA from the 73S class had a ratio of 47.5%. By comparison, chloroplast ribosomes of C. reinhardtii were found to sediment at 70S and contain rRNA molecules of 23S and 16S, with a G + C content of 51.0%. These findings support the concept that the Polytoma leucoplast possesses characteristic genetic and protein-forming systems.

RNA metabolism in HeLa cells at reduced temperature. I. Modified processing of 45S RNA.

Incubation of HeLa cells at suboptimal temperature has been used to study the synthesis of 45S ribosomal RNA precursor and the individual steps of the subsequent processing to 28S RNA. Below 20 degrees C no detectable 45S RNA is formed. The processing of 45S RNA to 32S RNA ceases around 15 degrees C, and the processing of 32S RNA to 28S RNA is inhibited near 25 degrees C. Prolonged incubation at reduced temperature results in further modification of the processing, resulting in the apparent accumulation of 41S RNA. The products of these reactions at reduced temperature appear normal in that the ribosomal RNA made at 27 degrees C can be isolated from functional polyribosomes in the cytoplasm after a short incubation at 37 degrees C.

Ribosomes bound to chloroplast membranes in Chlamydomonas reinhardtii.

The amount of chloroplast ribosomal RNAs of Chlamydomonas reinhardtii which sediment at 15,000 g is increased when cells are treated with chloramphenicol. Preparations of chloroplast membranes from chloramphenicol-treated cells contain more chloroplast ribosomal RNAs than preparations from untreated cells. The membranes from treated cells also contain more ribosome-like particles, some of which appear in polysome-like arrangements. About 50% of chloroplast ribosomes are released from membranes in vitro as subunits by 1 mM puromycin in 500 mM KCl. A portion of chloroplast ribosomal subunits is released by 500 mM KCl alone, a portion by 1 mM puromycin alone, and a portion by 1 mM puromycin in 500 mM KCl. Ribosomes are not released from isolated membranes by treatment with ribonuclease. Membranes in chloroplasts of chloramphenicol-treated cells show many ribosomes associated with membranes, some of which are present in polysome-like arrangements. This type of organization is less frequent in chloroplasts of untreated cells. Streptogramin, an inhibitor of initiation, prevents chloramphenicol from acting to permit isolation of membrane-bound ribosomes. Membrane-bound chloroplast ribosomes are probably a normal component of actively growing cells. The ability to isolate membrane-bound ribosomes from chloramphenicol-treated cells is probably due to chloramphenicol-prevented completion of nascent chains during harvesting of cells. Since chloroplasts synthesize some of their membrane proteins, and a portion of chloroplast ribosomes is bound to chloroplast membranes through nascent protein chains, it is suggested that the membrane-bound ribosomes are synthesizing membrane protein.

Mechanism of the cation effect in subfractionation of microsomes.

It was previously found that cations introduced into a discontinuous sucrose gradient exert a very pronounced effect on microsomal vesicles, and this principle proved to be effective in microsomal subfractionation. The mechanism of the cation effect was investigated. By using the radioactive isotopes (137)Cs and (85)Sr, it could be calculated that the amount of ions bound to the various subfractions increases their density by 0.14%, thereby enhancing the sedimentation velocity by only approximately 7%. In the presence of Cs(+) the total volume of the microsomal pellet was decreased by approximately 15%. Assuming this change in volume to be due to a contraction of the individual vesicles, a roughly 2(1/2)-fold increase in sedimentation velocity would be expected. It is further demonstrated, on the basis of light scattering and millipore filtration experiments, that monovalent cations cause an extensive aggregation of rough microsomes and a less pronounced aggregation of smooth microsomes. The mean radius of the sedimenting particles of rough microsomes was found to be at least doubled or trebled in the presence of Cs(+), which would give a 4- to 9-fold increase in the sedimentation velocity. Aggregation, therefore, appears to be the main factor in the accelerated sedimentation of rough microsomes in the presence of CsCl. Divalent cations exert a similar effect on a subfraction of the smooth microsomes. Isolated smooth microsomes are very unstable and often exhibit spontaneous aggregation. The presence of attached ribosomes, however, appears to impart greater stability to the rough microsomes as well as increasing their ability to bind monovalent cations. The primary cause of the aggregation of microsomal vesicles is probably due to a change in net charge.

Electron microscopy of the ammoniacal silver reaction for histones in the erythropoietic cells of the chick.

The product of the postformalin ammoniacal silver reaction, which has been claimed to distinguish lysine-rich from arginine-rich histones with the light microscope on the basis of a color difference, was examined in developing erythroid cells of chick bone marrow with the electron microscope. Stem cells and early erythroblasts exhibit no, or little, ammoniacal silver reaction product, while small basophilic erythroblasts, polychromatophilic erythrocytes, and reticulocytes exhibit an increasing amount of reaction product as maturation proceeds. The reaction product is in the form of discrete electron-opaque particles associated with heterochromatin. The ammoniacal silver reaction in the erythroid cell series is interpreted as reflecting either the accumulation of newly synthesized arginine-rich histones or changes in the availability of reactive sites in preformed histones.

Studies on the function of intracellular ribonucleases. IV. Some observations on the properties of ribosomes and polysomes from rat liver and hepatomas.

Polysome and ribosome preparations from normal rat liver and from a series of transplantable rat hepatomas of different growth rates were compared. All the hepatomas had a significantly higher percentage of RNA in a polysome preparation than did the normal liver, and the polysome preparations from the tumors, with the exception of the Dunning hepatoma which has a high lipid content, gave a greater yield of RNA and protein per gram of wet tissue than the liver did. Heavier polysomes were considerably less prevalent in the tumors than in the liver, and the tumors contained a larger proportion of monomer and dimer ribosomes than the liver did. Evidence is presented that the increased monomer and dimer ribosome population of the hepatomas studied is not an artifact of preparation, but represents the true intracellular distribution. Ribosomes from normal liver and Morris 5123-D hepatoma were readily dissociated by 20 min' treatment with 1.0 mM EDTA, but ribosomes from the Dunning, Novikoff ascites, and McCoy MDAB hepatomas were little affected by such treatment. With higher concentrations of EDTA, the ribosomes from the Novikoff ascites and McCoy MDAB hepatomas broke down and did not form specific subunits as did ribosomes from liver and the Morris 5123-D hepatoma but rather gave rise to a variety of small degradation products. This behavior is ascribed to a higher RNase content of the Novikoff and McCoy MDAB hepatomas. Dunning hepatoma ribosomes were resistant to 4 mM EDTA.

Membrane-bound ribosomes of myeloma cells. I. Preparation of free and membrane-bound ribosomal fractions. Assessment of the methods and properties of the ribosomes.

A cell fractionation procedure is described which allowed, by use of MOPC 21 (P3K) mouse plasmocytoma cells in culture, the separation of the cytoplasmic free and membrane-bound ribosomes in fractions devoid of mutual cross-contamination, and in which the polyribosomal structure was entirely preserved. This was achieved by sedimentation on a discontinuous sucrose density gradient in which the two ribosome populations migrate in opposite directions. A variety of controls (electron microscopy, labeling of membrane lipids, further repurification of the isolated fractions) provided no evidence of cross-contamination of these populations. However, when an excess of free 60S or 40S subunits, labeled with a different isotope, was added to the cytoplasmic extract before fractionation, the possibility of a small amount of trapping and/or adsorption of free ribosomal particles by the membrane fraction was detected, especially in the case of the 60S subunits; this could be entirely prevented by the use of sucrose gradients containing 0.15 M KC1. EDTA treatment of the membrane fraction detached almost all the 40S subunits, and about 70% of the 60S subunits. 0.5 M KC1 detached only 10% of the ribosomal particles, which consist of the native 60S subunits and the monoribosomes, i.e. the bound particles inactive in protein synthesis. Analysis in CsC1 buoyant density gradients of the free and membrane-bound polyribosomes and of their derived 60S and 40S ribosomal subunits showed that the free and membrane-bound ribosomal particles have similar densities.

Ultrastructural and biochemical studies on the precursor ribosomal particles isolated from rat liver nucleoli.

Sucrose density gradient analyses of pH 5.5 and pH 7.4 extracts from rat liver nucleoli revealed the presence of two broad peaks of approximately 60S and 80S, and 60S and 80-100S, respectively. Ribonucleoprotein (RNP) particles containing precursor ribosomal RNA in these peaks have been characterized by electron microscopy and RNA analyses. Spherical particles only were found in the 60S peak of the pH 5.5 extract, from which 28S RNA and smaller RNA (23S and 18S RNA) exclusively were extracted. In the broad 80S peak of the pH 5.5 extract, about 60% of the particles were spherical while 30% were rodlike. In the RNA species present there were 28S plus smaller RNA (80%) and 35S RNA (20%). The 60Speak of the pH 7.4 extract contained mainly spherical particles (84%), and the RNA species present was mostly 28S plus smaller RNA (89%). In addition to spherical particles (43%), a number of rodlike (31%) and filamentous molecules (26%) were observed in the heavier side of the 80-100S peak of the pH 7.4 extract, from which 45S (14%), 35S (26%), and 28S and smaller RNA (60%) were extracted. Thus the precursor ribosomal particles containing 45S RNA and 35S RNA appear to be filamentous and rodlike molecules, respectively. Folding of loose ribonucleoprotein filaments into compact, spherical, large subparticles may be part of the maturation process of ribosomal large subparticles, in addition to the so-called sequential cleavage of RNA.

Changes in the ribosome content, principal microsomal protein composition, and secretory character of mammary epithelial rough endoplasmic reticulum during differentiation. Evidence that messenger RNAs specific for milk proteins are incorporated into rough endoplasmic reticulum formed de novo after parturition.

The equilibrium density distribution, protein composition, and secretory character of mouse mammary epithelial rough microsomes have been determined during differentiation. The density range exhibited by the rough microsomes broadens during mammary development; rough microsomes within the 1.25-1.29 g/ml density range appear soon after conception and then within the 1.30-1.34 range after the onset of lactation. The appearance of these denser microsomes represents the progressive increase of the average ribosome content of the rough endoplasmic reticulum (ER) during gestation and lactation. Fractionation of rough microsomal proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that two proteins, having molecular weights of 57,000 and 76,000, occur to a significant extent only during lactation and are then most prominent in the very dense rough microsomes of the 1.30-1.34 range. Nascent polypeptide chains discharged (by incubation with puromycin) from 17-days lactation rough microsomes in either the 1.21-1.29 or 1.30-1.34 density range are distributed equally between the intra- and extravesicular compartments. Whereas 36% of the chains are discharged intravesicularly from 1-day lactation rough microsomes in the 1.30-1.34 range, only 25% are so discharged from those in the 1.21-1.29 range. The results indicate (a) that there is no correlation between the relative levels in lactation rough microsomes of the two microsomal proteins which become prominent during lactation and the extent of secretory activity and (b) that for a short period after parturition the rough ER elements bearing high surface densities of ribosomes have a greater proportion of ribosomes synthesizing milk proteins than the rough ER elements with moderate ribosome densities.

Composition of cellular membranes in the pancreas of the guinea pig. IV. Polyacrylamide gel electrophoresis and amino acid composition of membrane proteins.

TWO METHODS OF POLYACRYLAMIDE GEL ELECTROPHORESIS (THE ACID METHOD OF EYTAN AND OHAD AND THE NA DODECYLSULFATE (SDS) DISC METHOD OF MAIZEL) HAVE BEEN USED FOR ANALYZING THE PROTEINS OF GEL FRACTIONS ISOLATED FROM THE GUINEA PIG PANCREATIC EXOCRINE CELLS AND IN PARTICULAR THE PROTEINS BOUND TO THE MEMBRANES INVOLVED IN THE SYNTHESIS, INTRACELLULAR TRANSPORT, AND DISCHARGE OF SECRETORY ENZYMES: rough (RM) and smooth microsome (SM) membranes, zymogen granule (ZG) membranes, and plasma membranes (PM). Since in the two systems the electrophoretic mobility of proteins depends on different factors (size, shape, and net charge of molecules in the acid system; size only in the SDS system) a deeper insight into the protein composition of the fractions could be obtained. The gel patterns of RM, SM, and ZG membranes turned out to be accounted for mainly by segregated secretory enzymes (in rough microsomes also by ribosome proteins) and thus were found to share most of the bands. In contrast, with highly purified membrane fractions different patterns were obtained: RM and SM membrane proteins turn out to contain a large number of different proteins with molecular weights varying between approximately 150,000 and 15,000 daltons. The pattern of ZG membranes was greatly different in the two systems: only two bands were separated by the acid method and as many as 23 by the SDS method. PM gave a rather complex pattern in either system. Both ZG membranes and PM were found to contain a large proportion of low molecular weight proteins. Nothing appears in common between the proteins of SM membranes (primarily of Golgi origin) and those of ZG membranes, while the latter and PM exhibit a certain degree of similarity. By amino acid analysis we found only slight differences: relative to the other fractions: RM membranes were higher in basic amino acids and ZG membranes contained a larger amount of methionine. Taken together with recent data on lipid composition and enzyme activities of the same fractions, these results indicate that the membranes of the pancreatic exocrine cells are chemically and functionally distinct, and hence do not mix randomly with one another during the transport of secretory products.

Mitoribosomes from Candida utilis. Morphological, physical, and chemical characterization of the monomer form and of its subunits.

Highly purified mitochondrial ribosomes (mitoribosomes) have been obtained from the yeast Candida utilis. Sedimentation analysis in sucrose gradients made in 5 mM MgCl(2), 1 mM Tris, pH 7.4 and 50 mM KCl clearly distinguishes mitoribosomes (72S) from cytoplasmic ribosomes (cytoribosomes) (78S). Mitoribosomes are completely dissociated into 50S and 36S subunits at 10(-4)M MgCl(2) whereas complete dissociation of cytoribosomes into 61S and 37S subunits occurs only at 10(-6)M MgCl(2) Electron microscopy of negatively stained mitoribosomes (72S peak) shows bipartite profiles, about 265 x 210 x 200 A Characteristic views are interpreted as frontal, dorsal, and lateral projections of the particles, the latter is observed in two enantiomorphic forms Mitoribosome 50S subunits display rounded profiles bearing a conspicuous knoblike projection, reminiscent of the large bacterial subunit. The 36S subunits show a variety of angular profiles. Mitoribosomal subunits are subject to artifactual dimerization at high Mg(2+) concentration Under these conditions, a supplementary 80S peak arises. Electron microscopic observation of the 80S peak reveals closely paired particles of the 50S type Buoyant density determinations after glutaraldehyde fixation show a single peak at rho = 1.48 for mitoribosomes and 1.53 for cytoribosomes In the presence of ethylenediaminetetraacetate (EDTA), two species of RNA, 21S and 16S, are obtained from mitoribosomes, while 25S and 17S RNA are obtained from cytoribosomes It is established that the small and large RNA species are derived from the 36S and 50S subunits, respectively, by extraction of the RNA from each subunit The G + C content of the RNA is lower for mitoribosomes (33%) than for cytoribosomes (50%). Incubation of C utilis mitochondria with leucine-(14)C results in the labeling of 72S mitoribosomes. The leucine-(14)C incorporation is inhibited by chloramphenicol and resistant to cycloheximide Puromycin strips the incorporated radioactivity from the 72S mitoribosomes, which is consistent with the view that leucine-(14)C is incorporated into nascent polypeptide chains at the level of mitoribosomes

Stabilities of nuclear and messenger RNA molecules in sea urchin embryos.

The kinetics of accumulation of radioactive adenosine in adenosine triphosphate and in RNA of nuclear, cytoplasmic, and polysomal fractions of sea urchin embryos have been analyzed. 85% of the RNA synthesized decays in the nucleus with an apparently uniform half-life of about 7 min. The remaining 15% goes to the cytoplasm, mostly entering polysomes, and decays with a quite uniform half-life of about 75 min. The nuclear RNA accounts for one-third and the cytoplasmic RNA accounts for two-thirds of the total unstable RNA which accumulates at steady state in the embryo. The size distribution of short-labeled nuclear RNA is very similar to that of long-labeled messenger RNA, when both are extracted directly from the cells without a previous cell fractionation.

Ribosome crystallization in chicken embryos. I. Isolation, characterization, and in vitro activity of ribosome tetramers.

Isolated tetrameric particles (166S) derived from the crystalline lattices known to appear in hypothermic chicken embryos consist of mature 80S ribosomes which contain all species of ribosomal RNA and a complete set of ribosomal proteins. Ribosome tetramers are not a special type of polysomes since in solutions of high ionic strengths (500 mM KCl and 50 nM triethanolamine-HCl buffer) containing 5 mM MgCl(2) they dissociate into 40S and 60S ribosomal subunits, without the need of puromycin, and at a concentration of Mg(++) higher than 3 mM they are not disassembled by mild RNase treatment. Tetramers spontaneously disassemble into 80S monomers when the Mg(++) concentration is lowered to 1 mM at relatively low ionic strength. Tetramers failed to couple in vitro puromycin-(3)H into an acid-insoluble product, indicating the lack of nascent polypeptide chains. Although tetramers have no endogenous messenger RNA activity, they can be programmed in vitro with polyuridylic acid (poly U) to synthesize polyphenylalanine. All ribosomes within a tetramer can accept poly U, without the need of disassembly of the tetramers into monomers or subunits.