Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila,Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria

Ribosomal subunits of Caldariella acidophila (max.growth temp., 90°C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70°C) and Escherichia coli (max. growth temp., 47°C) with respect to (a) bihelical content of rRNA; (b) G·C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. 1. Subunits of C. acidophila ribosomes (T_m = 90–93°C) exhibit considerable thermal tolerance over their B. acidocaldarius (T_m = 77°C) and E. coli counterparts (T_m = 72°C). 2. Based on the ‘melting’ hyperchromicities of the intact ribosomal subunits a 51–55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67–70%, appears to be involved in hydrogen bonding in the naked rRNA species. 3. The G·C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63–64% G·C, compared to 58.5% G·C for B. acidocaldarius and 55% G·C for E. coli. 4. The increment in ribosome T_m values with increasing thermophily is greater than the increase in T_m for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). 5. The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release rRNA from Celite-bound E. coli ribosomes. 6. Compared to E. coli, the C. acidophila 50 and 30 S ribosomal subunits are considerably less susceptible to treatment designed to promote ribosome unfolding through depletion of magnesium ions.     

Minor nucleosides in RNA: optical studies of dinucleoside phosphates containing inosine

From the study of circular dichroism (CD) spectra and hypochromism we conclude that the dinucleoside phosphates IpA, ApI, and IpI stack, while IpU stacks very little. Studies with various concentrations of IpI in low salt, and 1M NaCl indicate that the stacking geometry of this compound is sensitive to the ionic strength of solution. The CD of poly I is presented and compared to the data for IpI. Little change was found in the CD of poly (G,I) (1 : 1) with change of salt concentration, and we conclude that, unlike Poly I, there is no major structural change. From the CD of poly (G, I), IpI, and GpG, the CD of IpG plus GpI is calculated by using the nearest-neighbor approximation. From the calculated spectrum, we tentatively conclude that there is stacking in either IpG or GpI or both.     

Minor nucleosides in RNA: Optical studies of dinucleoside phosphates containing dihydrouridine

Photoreduction with NaBH₄ was used to reduce the dinucleoside phosphates ApU, UpA, and GpU to the corresponding molecules containing dihydrouridine (H). Also obtained from this reaction are dinucleoside phosphates containing (β-N-ribosyl) ureido-propanol, an open ring form of dihydrouridine. The results of CD and ultraviolet absorption studies with these compounds imply that HpA is strongly stacked, but that ApH and GpH are only slightly stacked. The temperature dependence of the CD suggests that the stacking in HpA is unusual. The results with compounds containing open ring forms of dihydrouridine indicate that there is more intramolecular interaction when the ring is open than when it is closed.     

Endocytosis and the adaptive acid hydrolases in Tetrahymena pyriformis GL

Endocytosis of yeast cells by Tetrahymena pyriformis GL for a period of 2.5 h produced changes in cellular acid hydrolases. Acid phosphatase, acid deoxyribonuclease and acid proteinase activities were markedly increased, whereas there was a decrease in acid ribonuclease activity and little change in -glucosidase activity. These alterations do not appear to be due to any alteration in the rates of secretion of these enzymes into the milieu. Evidence is presented that the cellular enzyme increases found upon endocytosis of yeast reflect changes in lysosomal enzymes, because it was shown that the acid phosphatase activity increase resulted in an increased amount of latent enzyme within the cell. The results also support the idea that there are at least 3 distinct populations of lysosomes, in addition to phagolysosomes, present in Tetrahymena pyriformis GL, with different modes of formation. There appears to be a large excess of lysosomes, uncombined with phagosomes, present in these fed cells since latency averaged 66% in broken-cell preparations which contained very few intact phagolysosomes. The phagolysosomal acid phophatase activity cannot account for more than 34% of that present in the cell. The endocytosis of yeast in the presence of growth medium resulted in a marked drop in the rate of cell division as compared to cells growing in the growth medium alone. The results are discussed.     

‘Milk’ secretion for embryogenesis in a viviparous cockroach

The brood sac of viviparous Diploptera punctata is a typical insect integumentary gland which secretes a ‘milk’ containing protein and carbohydrate to nourish the developing embryos. During gestation the secretory cells proliferate organelles of protein synthesis and secretion and brood sac wet weight, protein content, synthetic activity and secretory output increase five- to six-fold ; a maximum of 0.4 mg protein was collected in 24 hr from one brood sac in a later stage of gestation. Following parturition, when secretory activity ceases, these parameters fall markedly, and the secretory cells decrease their mass by autophagic regression. Acid phosphatase has been located histochemically in autolysomes and assayed in brood sac homogenates; activity reaches a maximum five days after parturition.     

An Improved Diluent for Rubella Hemagglutination and Hemagglutination-Inhibition Tests

Rubella hemagglutinating (HA) antigen was prepared in BHK-21 tissue as 5% cell suspensions and from unconcentrated and 20× concentrated infected supernatant fluids. In some instances, unconcentrated fluids were treated with Tween 80 and ether; cell suspensions were treated with ether alone. Preparations were tested for HA activity in dextrose-gelatin-Veronal (DGV) buffer solutions; 0.85% NaCl; Sorenson's phosphate-buffered saline, pH 7.2; and a diluent of 0.9% NaCl, 0.1% CaCl₂ (anhydrous), and 0.1% MgSO₄·7H₂O. HA titers were consistently two- to fourfold higher in the saline with added Ca⁺⁺ and Mg⁺⁺ than in DGV. Hemagglutination-inhibition titers of paired human sera were the same in either diluent. It is suggested that the interaction between rubella HA antigen and the red cells of young (less than 1-day-old) chicks may be at least partially ion dependent and that titers are enhanced by increased quantities of divalent cations.     

Calculated optical properties of 64 trinucleoside diphosphates

The optical rotatory dispersion and ultraviolet absorption spectra of the 64 trinucleoside diphosphates containing the bases A, U, C, and G have been calculated by using a simple semiempirical approach. These calculations accurately predict the optical properties of the nine trimers for which extensive experimental results are available. The computed optical data should be useful in the identification of oligonucleotides obtained in the course of sequence determination of ribonucleic acids and should simplify the determination of the concentration of oligonucleotides in aqueous solution. Additional calculations indicate that it should be possible to analyze most, mixtures of sequence isomers of trinucleoside diphosphates by direct, measurement of the ORD of the mixture at neutral pH.     

RNA pseudoknots. Stability and loop size requirements.

The effects of ionic conditions, loop size and loop sequence on the formation of pseudoknots by RNA oligonucleotides have been investigated using biochemical and biophysical methods. An oligonucleotide with the sequence 5' GCGAUUUCUGACCGCUUUUUUGUCAG 3' and oligonucleotides with variations in the sequences of the two loop regions, denoted by bold face type, were studied. Each sequence with the potential to form a pseudoknot can also form two stable hairpins. The pseudoknot structure is stabilized relative to the hairpins by addition of Mg2+. Even in the presence of Mg2+, the pseudoknots formed by the sequences investigated are only marginally more stable (1.5 to 2 kcal mol-1 in free energy at 37 degrees C) than either of the constituent hairpins. The pseudoknot structure is the stable conformation in the presence of Mg2+ when the first loop region is at least three nucleotides and the second is at least four nucleotides. Further deletion of nucleotides from the loop regions stabilizes possible hairpin structures relative to the pseudoknot and equilibria among secondary and tertiary structures result.