Cytogenetic analysis of oocytes and early preimplantation embryos from XO mice.

The cytoplasm of early sea urchin embryos contains nonribosomal, high molecular weight RNA both associated with ribosomes in polysomes and free of ribosomes in particles termed free RNP. In a 1-hr labeling period, 50% of the newly synthesized RNA enters the pool of ribosome-free RNP particles during the cleavage stages, and this percentage decreases until less than 20% of the new RNA in the mesenchyme blastula stage is found in the free RNP. mRNA from both polysomes and free RNP contain poly(A)(+) and poly(A)(?) species. During the cleavage stages only 8–10% of the RNA from each fraction is polyadenylated; however, in the blastula, 40–50% of the nonhistone polysomal RNA is polyadenylated while only 22–30% of the free RNP RNA is polyadenylated. At any developmental stage, the poly(A)(+)RNA from the free RNA and polysomes have identical sedimentation profiles; this is also the case for the poly(A)(?)RNA except for the absence of the 9 S histone mRNA from the free RNP. Changes in poly(A)(+)RNA content and sedimentation profiles during development occur simultaneously in the free RNP and the polysomes. Kinetic studies of these two RNP populations as well as nuclear RNP show that the bulk of the free RNP are not unusually stable cytoplasmic components. The free RNP decay with a half-life of about 40 min while nuclear RNA and polysomal RNA display half-lives of about 12 and 65 min, respectively. Further, the rate of synthesis of the free RNP is not consistent with their being the only precursors for polysomes. Our estimates of the rates of synthesis for nuclear RNA, polysomes, and free RNP are, respectively, 1.1 × 10^?15, 2.2 × 10^?16, and 5.0 × 15^?17 g/min/nucleus. The data on free RNP is discussed in terms of translational regulation of protein synthesis in the developing sea urchin.     

A Cytochemical Study on the Pancreas of the Guinea Pig : IV. Chemical and Metabolic Investigation of the Ribonucleoprotein Particles

Five ribonucleoprotein (RNP) fractions were isolated from the postmitochondrial supernatant of the pancreas of the guinea pig. Two were obtained from the microsomes which, by deoxycholate (DOC) treatment, were subdivided into a DOC-soluble and a DOC-insoluble fraction. The latter was taken to represent attached RNP particles. Two other fractions obtained from the microsomal supernatant supposedly represent free RNP particles existing as such in the cytoplasm, while a third fraction resisted sedimentation for 20 hours at 105,000 g and is considered to be a soluble nucleoprotein. These fractions exhibited different RNA/protein ratios and also different RNA turnover patterns, as determined after in vivo labelling with adenine-8-C¹⁴. However, little discernible differences could be detected in the nucleotide composition of the RNA moieties of these RNP fractions. Amino acid-"activating" enzymes were found to occur in the fraction containing the soluble nucleoproteins. The discussion focuses on the relationships between these fractions and protein synthesis in the pancreas, using data given in this and a previous paper, and data contained in the literature.     

Proteins associated with heterogeneous nuclear RNA in eukaryotic cells

When HeLa cell nuclei axe mechanically disrupted in either hypotonic or isotonic buffers, heterogeneous nuclear RNA is recovered from the post-nucleolar fraction in the form of EDTA-resistant ribonucleoprotein particles, which sediment between 40 S and 250 S in sucrose gradients containing 0.01 m or 0.15 m-NaCl. That the RNA in these particles is HnRNA² is indicated by its heterodisperse sedimentation (20 to 80 S) and its continued synthesis in concentrations of actinomycin D that selectively inhibit the synthesis of ribosomal RNA. The specificity of the HnRNA-protein complexes is evidenced by the failure of deliberate attempts to generate artificial RNP by the addition of deproteinized HnRNA to intact or disrupted nuclei at low ionic strength.The proteins bound to HnRNA are complex. In HeLa cells, HnRNP particles contain proteins with molecular weights from 39,000 to approximately 180,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric points between 4.9 and 8.3 (analytical isoelectric focusing). They are readily distinguishable from proteins in other cell fractions, including those in chromatin.Exposure of HeLa HnRNP particles to 0.5 m-NaCl reduces their average sedimentation velocity by approximately 30%. CsCl density-gradient analysis reveals that this is accompanied by the loss of a major portion of the proteins. However, a significant fraction of the HnRNP (25 to 30%) is resistant to high salt concentrations and continues to band at the same density as native HnRNP (1.43 g/cm³). This is true even after prolonged exposure (24 h) to high salt. The salt-resistant HnRNP is enriched for proteins above 60,000 molecular weight. In at least these two respects, this sub-class of HnRNP resembles “messenger RNP” prepared from cytoplasmic polyribosomes, which is also salt-stable and contains relatively high molecular weight proteins.HnRNP particles can also be recovered from HeLa cell nuclei lysed in high salt but these contain many extra proteins, notably histones, and sediment much faster in sucrose gradients than particles prepared as above. HnRNP is not liberated by extracting HeLa nuclei in 0.14 m-NaCl, pH 8.0 (Samarina et al., 1967) unless the temperature is 20 °C or higher. In this case the particles are converted to 45 S structures, which contain partially degraded HnRNA. 45 S particles can also be produced by subjecting 40 to 250 S HnRNP to a very limited digestion with pancreatic ribonuclease (1 to 2 hits/molecule).HnRNP particles have similar sedimentation velocities (40 to 300 S) when isolated under physiological ionic conditions from a variety of mammalian cells, including WI38 human diploid fibroblasts, mouse L-cells, monkey kidney cells and rat liver. However, electrophoresis reveals a distinct pattern of HnRNP proteins for each cell type. It is proposed that this cell-specificity reflects a situation in which HnRNA molecules that differ in nucleotide sequence are complexed with different sets of proteins, so that the resulting HnRNP particles are biochemically distinct at each genetic locus. This hypothesis is discussed in relation to the cytology of lampbrush and polytene chromosomes.     

A cytochemical study on the pancreas of the guinea pig. IV. Chemical and metabolic investigation of the ribonucleoprotein particles

Five ribonucleoprotein (RNP) fractions were isolated from the postmitochondrial supernatant of the pancreas of the guinea pig. Two were obtained from the microsomes which, by deoxycholate (DOC) treatment, were subdivided into a DOC-soluble and a DOC-insoluble fraction. The latter was taken to represent attached RNP particles. Two other fractions obtained from the microsomal supernatant supposedly represent free RNP particles existing as such in the cytoplasm, while a third fraction resisted sedimentation for 20 hours at 105,000 g and is considered to be a soluble nucleoprotein. These fractions exhibited different RNA/protein ratios and also different RNA turnover patterns, as determined after in vivo labelling with adenine-8-C(14). However, little discernible differences could be detected in the nucleotide composition of the RNA moieties of these RNP fractions. Amino acid-"activating" enzymes were found to occur in the fraction containing the soluble nucleoproteins. The discussion focuses on the relationships between these fractions and protein synthesis in the pancreas, using data given in this and a previous paper, and data contained in the literature.     

CHARACTERIZATION OF BRAIN RIBONUCLEOPROTEIN PARTICLES

Abstract— Brain RNP particles were characterized to determine whether they play a role in the regulation of brain protein synthesis. RNP particles were isolated from the postribosomal supernatant of cerebral hemispheres of young rabbits, employing conditions which minimize adventitious protein-RNA interactions. Brain RNP particles consist of a different set of proteins compared to proteins associated with either 40 and 60s ribosomal subunits or polysomal mRNA. Poly(A+)mRNA from brain RNP particles stimulates the incorporation of [³⁵S]methionine in a wheat embryo cell-free system and codes for a different set of proteins compared to poly(A+)mRNA isolated from polysomes (with some overlap; i.e. mRNA coding for brain-specific S100 protein is present in both RNP particles and polysomes).
Addition of total brain RNP particles to a cell-free wheat embryo system inhibits the endogenous incorporation of [³⁵S]methionine. Total RNP particles were fractionated by sucrose density gradient centrifugation into a'light'and a'heavy'fraction. The light RNP fraction inhibited while the heavy RNP fraction stimulated protein synthesis in the wheat embryo cell-free system. Analysis of the protein composition of fractionated RNP particles revealed that the light and heavy RNP particles contained different sets of proteins. Together these results suggested that one class of brain RNP particles may contain a translational inhibitor and may be involved in the regulation of protein synthesis in the brain.     

Characterization of proteins associated with nuclear ribonucleoprotein particles by two-dimensional polyacrylamide gel electrophoresis.

Rat liver nuclear ribonucleoprotein particles were prepared by two different methods and defined as 40S ribonucleoprotein (40S RNP) and heterogeneous nuclear ribonucleoprotein (HnRNP) particles. The RNP particles were either solubilized in 8 M urea--6 mM 2-mercaptoethanol--20 mM glycine--20 mM Tris--HCl (pH 8.4) or subjected to removal of RNA by phenol extraction prior to solubilizing the proteins in the urea buffer. The proteins associated with 40S RNP and HnRNP were heterogeneous and very similar in their electrophoretic patterns when analyzed by two-dimensional PAGE, except a protein with molecular weight of 62 000 and an isoelectric point (pI) of 6.2 was present only in HnRNP particles. At least 12 major and 22 minor components could be identified in both preparations. The major proteins were found at pI values varying from 6.0 to 8.5 and with molecular weights from 32 000 to 42 000, and a group of proteins with molecular weight approximately 65 000 were more prominent in HnRNP than in 40S RNP. The other components were found mainly at pI ranges from 5.0 to 6.5 with molecular weights from 43 000 to 65 000. The phenol method extracted essentially all proteins associated with either 40S RNP and HnRNP, but was less effective in extracting a group of proteins with pI values from 5.0 to 5.5 and more efficient for proteins with pI values from 7.5 to 8.5. When chromatin proteins isolated by phenol extraction were compared with HnRNP particle proteins isolated by the same method, the electrophoretic mobilities of the HnRNP particle proteins were found to be identical with a fraction nonhistone chromatin proteins. The 40S RNP particles were further purified by metrizamide isopycnic density gradient centrifugation. The electrophoretic patterns of these proteins were very similar to those prepared by sucrose density gradient centrifugation. Therefore, we concluded that the proteins of RNP particles constituted part of the chromatin proteins.     

Isolation of Ribonucleic Acids Having Template Activities from Particulate Components of Soybean Seeds

Ribonucleic acids having template activities were obtained from particulate components prepared from the postribosomal supernatant of soybean seeds. These RNA were 9 S and 18 S in size, and these corresponded to the components (9 S, 18 S) of high molecular weight RNA (H–RNA) prepared from the supernatant of 100,000×g centrifugation. The sizes of the particulate components were 37 S and 59 S, respectively. Larger particles contained 18 S and 9S RNA, and smaller particles contained 9S RNA, but not 18 S RNA. Those particulate components differed in absorption pattern and in the behaviour on sucrose gradient centrifugation depending on the concentration of Mg²⁷⁺ from the subunits of ribosomes.     

Globin messenger sequences in nuclear ribonucleoprotein particles of avian erythroblasts

Nuclear ribonucleoprotein particles were isolated from chick erythroblast nuclei. The particles were found to sediment as heterogeneous material. The major fraction of the rapidly synthesized RNP sedimented at 30 S, whereas the nuclei were found to contain a major, apparently more stable, RNP component sedimenting at about 40 S. The RNA isolated from the RNP particles was assayed for globin messenger activity in a wheat germ cell-free system. RNP sedimenting at relatively low S values (approx. 15 S) as well as RNP-particles of larger size code for globin. In addition to globin, the RNA of the particles codes also for other, not yet identified, proteins.     

Properties of broad bean yellow band virus, a possible new tobravirus

A virus was transmitted from broad bean plants in Apulia (Southern Italy) with leaves showing yellow rings, line patterns or yellow vein banding and malformations and necrosis of pods. Symptoms in some, but not all, test plants were similar to those induced by tobraviruses. Purified virus preparations contained two classes of rod-shaped particles containing c. 5% nucleic acid with sedimentation coefficients of 186S and 276S. After centrifugation to equilibrium in CsCl gradients, two components were resolved, with buoyant densities of 1·298 and 1·316 g/cm³. Unfractionated virus preparations contained two species of single-stranded RNA with mol. wts of c. 1·06 × 10⁶ and 2·48 × 10⁶ and one species of coat protein with mol. wt of c. 21 300. The modal lengths of the two classes of particles, both in plant sap and in purified preparations, were 77 nm (S particles) and 202 nm (L particles). L particles accumulated in infected cells in paracrystalline aggregates, whereas S particles were randomly distributed in the cytoplasm of cells. The virus was serologically unrelated to two isolates of tobacco rattle virus and two isolates of pea early-browning virus. The virus, named broad bean yellow band, is considered a distinct tobravirus.     

Thermal Regime and Stability of Jurumirim Reservoir (Paranapanema River,São Paulo,Brazil)

Thermal regime and stability in Jurumirim Reservoir (São Paulo, Brazil) were studied for a year. Isothermy and thermal stratification were observed from June to September and from October to March, respectively. The annual heat budget was 14.0 Kcal · cm⁻² and average heat gain per day was 67.2 cal · cm⁻² · day⁻¹. Maximum stability and wind work reached 324.2 and 3,037.5 g · cm · cm⁻², respectively. According to thermal structure and heat content, Jurumirim Reservoir was classified as a warm tropical monomictic lake.     

Identification of proteins functionally altered by chemical modification of the transfer RNA and polyuridylic acid binding sites of 30 S ribosomal subunits

Previous studies have shown that Rose Bengal-sensitized photo-oxidation of 30 S ribosomal subunits causes inactivation of tRNA binding and partial loss of poly(U) binding activities (Noller et al., 1971). The present studies, reconstitution of 30 S subunits from 16 S RNA, total protein from modified subunits, and purified proteins from untreated subunits, show that proteins S2 and S3 together completely restore these activities to the reconstituted subunits. The modified proteins are capable of in vitro assembly, and give rise to particles with normal sedimentation constants, showing that restoration of activity is not simply due to correction of an assembly defect.Protein S3 restores poly(U) binding and tRNA binding to the same extent, accounting for the lowered mRNA binding activity of the modified particles as well as a corresponding fraction of the tRNA binding activity. Protein S2 restores the remaining fraction of the tRNA binding activity, but has no effect on poly (U) binding. In 50 S-stimulated tRNA binding, proteins S1 and S5 are required in addition to S2 and S3 for full activity.     

Multiple states of U3 RNA in Novikoff hepatoma nucleoli

U3 RNA, a capped small nuclear RNA found thus far only in the nucleolus, has been implicated in the processing and/or transport of preribosomal RNA [Busch, H., Reddy, R., Rothblum, L., & Choi, Y. C. (1982) Annu. Rev. Biochem. 51, 617-654]. Tris(hydroxymethyl)aminomethane (Tris) (10 mM, pH 7.0) extracts of Novikoff hepatoma nucleoli, which contained about 80% of total nucleolar U3 RNA, were analyzed by sucrose density gradient centrifugation. Approximately 65% of the U3 RNA was bound to greater than 60S preribosomal ribonucleoprotein (RNP) particles, and about 15% sedimented at less than 20 S. The association between the 65% of U3 RNA that was bound to the preribosomal RNP particles was stable up to 55 degrees C. About 10% of U3 RNA was base paired to preribosomal RNA after deproteinization at 22 degrees C. The base-paired fraction of U3 RNA was released from the preribosomal RNA by heating to 45 degrees C or treating with 4 M urea. These results show that of the total nucleolar U3 RNP, (a) about 55% is bound to preribosomal RNP particles primarily by protein interactions, (b) about 10% is base paired to preribosomal RNA, (c) approximately 15% sedimented slowly and consisted presumably of free U3 RNP particles, and (d) the remaining 20% of U3 RNP was not extractable using 10 mM Tris buffer. On the basis of the different association states of U3 RNP particles, a model is proposed for the binding and dissociation events which take place between U3 RNP and preribosomal RNP particles.     

Influence of planting geometry on photosynthetically active radiation interception and dry matter production relationships in pearl millet

A field experiment was conducted at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) Center, Patancheru, India to study photosynthetically active radiation (PAR) interception and dry matter production relationships in pearl millet (Pennisetum americanum (L.) Leeke). Two pearl millet genotypes, BJ 104 (G₁) and ICH 226 (G₂) were sown at three planting geometries obtained by using combinations of row and plant spacings (S₁: 37·5 cm × 26·6 cm; S₂: 75·0 cm × 13·3 cm; S₃: 150·0 cm × 6·6 cm) such that plant population was constant at 100 000 ha⁻¹ in all treatments. Cumulative intercepted PAR was maximum (330 MJ m⁻²) in G₂S₂ and minimum (268 MJ m⁻²) in G₁S₃. Conversion efficiency values ranged from 1·87 g MJ⁻¹ in G₁S₂ to 2·32 g MJ⁻¹ in G₂S₃. Final above-ground dry matter followed the pattern of cumulative intercepted PAR and maximum dry matter (7·22 Mg ha⁻¹) was produced by G₂S₂ while G₁S₃ produced minimum dry matter (4·97 Mg ha⁻¹).     

On the synthesis of viral ribonucleic acids and ribonucleoproteins in the submitochondrial system completely free of interfering cytoplasmic contaminations

Summary The synthesis of virus-specific macromolecules was studied in the reconstituted system containing inner membrane-matrix fraction from rat liver mitochondria and infectious RNA of Venezuelian equine encephalomyelitis (VEE) virus. In a series of preliminary experiments it was shown that isolated submitochondrial fraction was completely free of interfering cytoplasmic contaminations and particularly, of cytoplasmic 80S ribosomes. VEE RNA when added to submitochondrial system caused significant stimulation of RNA and protein synthesis. These processes were resistant to actinomycin D which inhibited profoundly the synthesis of proper mitochondrial macromolecules. The stimulating effect of VEE RNA in experiments with submitochondrial system was about three times higher than that with intact mitochondria. The stimulation of¹⁴C-amino acid incorporation increased as a function of incubation time; a certain lag-period being observed. The newly formed virus-specific RNA's and ribonucleoproteins were identified with the aid of sedimentation analysis. In particular, radioactive RNA's with sedimentation coefficients 40S and 26-18S were isolated from the incubated system. These RNA's are similar respectively to VEE genome RNA and doublestranded VEE replicative RNA. In double labelling experiments with³H-uridine and¹⁴Camino acids it was shown that VEE RNA induced synthesis of ribonucleoproteins containing newly formed RNA and protein. These RNP possessed sedimentation coefficients 60-80S, 140S and 300S in sucrose gradient and buoyant densities 1.32 and 1.50 g/cm³ in cesium chloride gradients. These properties of ribonucleoproteins synthesized de novo in submitochondrial system are close to those of RNP intermediates of VEE virus reproduction in the infected cells. We concluded that viral RNA could program virus-specific synthesis in the submitochondrial system under conditions that eliminated the contribution of cytoplasmic ribosomes.     

Physicochemical studies on xylinan (acetan). III. Hydrodynamic characterization by analytical ultracentrifugation and dynamic light scattering

A laboratory-made sample of the polysaccharide xylinan (acetan) has been further characterized with respect to (i) purity, (ii) molar mass and polydispersity, and (iii) gross conformation by a combination of hydrodynamic measurements (sedimentation velocity and equilibrium analytical ultracentrifugation, viscometry, and dynamic light scattering) in aqueous NaCl (I = 0.10 mol·L⁻¹). Sedimentation velocity diagrams recorded using Schlieren optics revealed highly pure material sedimenting as a single boundary [s^o_20.w = 9.5 ± 0.7) S; k_s = (273 ± 112) mL/g]. The hypersharp nature of these boundaries is symptomatic of a polydisperse and highly nonideal (in the thermodynamic sense) system. Low speed sedimentation equilibrium in the analytical ultracentrifuge using Rayleigh interference optics and two different types of extrapolation procedure (involving point and whole-cell molar masses) gave a weight average molar mass M_w of (2.5 ± 0.5) × 10⁻⁶ g·mol⁻¹ and also a second virial coefficient, B = (2.8 ± 0.7) × 10⁻⁴ mL·mol·g⁻², both values in good agreement with those from light scattering-based procedures (Part II of this series). A dynamic Zimm plot from dynamic light scattering measurements gave a z-average translational diffusion coefficient D^o_20.w = (3.02 ± 0.05) × 10⁻⁸ cm²·s⁻¹ and the concentration-dependence parameter k_D = (370 ± 15) mL/g. Combination of s^o_20.w with D^o_20.w via the Svedberg equation gave another estimate for M_w of ≅ 2.4 × 10⁶ g/mol, again in good agreement. Both the Wales-van Holde ratio (k_s/[η]) ≅ 0.4 (with [η] = (760 ± 77) mL/g) and the ρ-parameter (ratio of the radius of gyration from static light scattering to the hydrodynamic radius from dynamic light scattering) as ρ > 2.0 all indicate an extended conformation for the macromolecules in solution. These findings, plus Rinde-type simulations of the sedimentation equilibrium data are all consistent with the interpretation in terms of a unimodal wormlike coil model performed earlier. © 1996 John Wiley & Sons, Inc.     

Characterization of a native mRNA containing preinitiation complex from rabbit reticulocytes: RNA and protein constituents

From a rabbit reticulocyte postpolysomal supernatant a fraction has been isolated which is enriched in ribosomal particles sedimenting at 50S. This fraction is efficiently in vitro translated predominantly into α-globin. Besides the RNAs and proteins of the small ribosomal subunit the 50S particle contains α-globin mRNA and additional high molecular weight proteins, most of which correspond to polypeptides of the initiation factors eIF-2 and eIF-3. The 50S particle may represent a native [mRNA·40S·eIF′s·Met-tRNA_f·GTP] complex which may occur in vivo as a translatable intermediate in the initiation sequence.     

The ribosomal binding site for eukaryotic elongation factor EF-2 contains 5 S ribosomal RNA

The possible location of RNA in the ribosomal attachment site for the eukaryotic elongation factor EF-2 was analysed. Stable EF-2 · ribosome complexes formed in the presence of the non-hydrolysable GTP analogue GuoPP[CH₂]P were cross-linked with the short (4 Å between the reactive groups) bifunctional reagent, diepoxybutane. Non-cross-linked EF-2 was removed and the covalent factor-ribosome complex isolated. No interaction between EF-2 and 18 S or 28 S rRNA could be demonstrated. However, density gradient centrifugation of the cross-linked ribosomal complexes showed an increased density (1.25 g/cm³) of the factor, as expected from a covalent complex between EF-2 and a low-molecular-weight RNA species. Treatment of the covalent ribosome-factor complexes with EDTA released approx 50% of the cross-linked EF-2 from the ribosome together with the 5 S rRNA · protein L5 complex. Furthermore, the complex co-migrated with the 5S rRNA · L5 particle in sucrose gradients. Polyacrylamide gel electrophoresis showed that EF-2 was directly linked to 5 S rRNA in the 5 S rRNA · L5 complex, as well as in the complexes isolated by density gradient centrifugation. No traces of 5.8 S rRNA or tRNA could be demonstrated. The data indicate that the ribosomal binding domain for EF-2 contains the 5 S rRNA · protein L5 particle and that EF-2 is located in close proximity to 5 S rRNA within the EF-2 · GuoPP[CH₂]P · ribosome complex.