Codon-anticodon interaction at the ribosomal E site

The question of whether or not the tRNA at the third ribosomal binding site specific for deacylated tRNA (E site) undergoes codon-anticodon interaction was analyzed as follows. Poly(U)-programmed ribosomes each carrying two [14C]tRNAPhe molecules were subjected to a chasing experiment using various tRNA species. At 0 degree C Ac[3H]Phe-tRNAPhe did not trigger any chasing whereas deacylated cognate tRNAPhe provoked a strong effect; non-cognate tRNALys was totally ineffective. This indicates that the second [14C]tRNAPhe cannot be present at the A site but rather at the E site (confirming previous observations). In the presence of poly(U) or poly(A) ribosomes bound the cognate tRNA practically exclusively as second deacylated tRNA, i.e. [14C]tRNAPhe and [14C]tRNALys, respectively. Thus, the second deacylated tRNA binds in a codon-dependent manner. [14C]tRNALys at the P site and Ac[3H]Lys-tRNALys at the A site of poly(A)-primed ribosomes were translocated to the E and P sites, respectively, by means of elongation factor G. The E site-bound [14C]tRNALys could be significantly chased by cognate tRNALys but not by non-cognate tRNAPhe, indicating the coded nature of the E site binding. Additional evidence is presented that the ribosome accommodates two adjacent codon-anticodon interactions at either A and P or P and E sites.     

Characterization and in vitro translation of polyadenylated messenger ribonucleic acid from Neurospora crassa.

Ribonucleic acid (RNA) extracted from Neurospora crassa has been fractionated by oligodeoxythymidylic acid [oligo(dT)]-cellulose chromatography into polyadenylated messenger RNA [poly(A) mRNA] and unbound RNA. The poly(A) mRNA, which comprises approximately 1.7% of the total cellular RNA, was further characterized by Sepharose 4B chromatography and polyacrylamide gel electrophoresis. Both techniques showed that the poly(A) mRNA was heterodisperse in size, with an average molecular weight similar to that of 17S ribosomal RNA (rRNA). The poly(A) segments isolated from the poly(A) mRNA were relatively short, with three major size classes of 30, 55, and 70 nucleotides. Gel electrophoresis of the non-poly(A) RNA indicated that it contained primarily rRNA and 4S RNA. The optimal conditions were determined for the translation of Neurospora mRNA in a cell-free wheat germ protein-synthesizing system. Poly(A) mRNA stimulated the incorporation of [14C]leucine into polypeptides ranging in size from 10,000 to 100,000 daltons. The RNA that did not bind to oligo(dT)-cellulose also stimulated the incorporation of [14C]leucine, indicating that this fraction contains a significant concentration of mRNA which has either no poly(A) or very short poly(A) segments. In addition, the translation of both poly(A) mRNA and unbound mRNA was inhibited by 7-methylguanosine-5'-monophosphate (m7G5'p). This is preliminary evidence for the existence of a 5'-RNA "cap" on Neurospora mRNA.     

Growth responses of ciliate protozoa to the abundance of their bacterial prey

The growth rate or numerical response of five species of bactivorous ciliates to the abundance ofEnterobacter aerogenes was examined in monoxenic culture. The ciliatesColpidium campylum, C. colpoda, Glaucoma scintillons, G. frontata, andCyclidium glaucoma were isolated from a small pond. Four were grown in shaken cultures, while three were grown in cultures in which the bacteria were allowed to settle on the bottom of the culture vessel. Of the seven response curves generated, four had distinct thresholds, so that the Michaelis-Menten model usually fitted to ciliate numerical response curves was not appropriate. In shaken cultures, half-saturation prey densities ranged from 5.5 × 10⁶ to 42.9 × 10⁶ bacteria/ml. In unshaken cultures, half-saturation densities ranged from 0.057 × 10⁶ to 14.6 × 10⁶ bacteria/cm². Two species grown on both suspended and settled bacteria attained higher growth rates and had lower half-saturation prey densities feeding on settled bacteria.     

Neurospora crassa mitochondrial transfer RNAs.

Total mitochondrial tRNA from Neurospora crassa was characterized by base composition analysis, one- and two-dimensional gel electrophoreses and reversed-phase chromatography on RPC5. The guanosine + cytidine content was about 43%, as compared to 60% for cytoplasmic tRNA. The modified nucleoside content was low and about the same as that of total yeast mitochondrial tRNA, though the G + C content is very different. We found psi, T, hU, t6A, m1G, M2G, m22G. Neither the eukaryotic "Y" base, nor the prokaryotic s4U were present. On two-dimensional polyacrylamide gel electropherograms about 25 species were separated. One species for phenylalanine, two for leucine and two for methionine could be located. Neurospora crassa mitochondrial tRNA does not hybridize with yeast mitochondrial DNA.     

The translocation inhibitor tuberactinomycin binds to nucleic acids and blocks the in vitro assembly of 50S subunits.

Binding studies were performed with a [14C]-labelled derivative of viomycin, tuberactinomycin 0 (TUM O). TUM O bound to 30S and 50S subunits. The binding component was the RNA, since ribosomal proteins did not bind the drug. Other RNAs such as tRNA, phage RNA (MS2), and homopolynucleotides also bound the drug. Striking differences in the binding capacity of the various homopolynucleotides were found. Poly(U) bound strongly, poly(G) and poly(C) bound intermediately, whereas poly(A) showed a very low binding. DNA also bound TUM O, although with native DNA the binding was only weak. Finally the effects of viomycin on the assembly in vitro of the 50S subunit from E. coli were tested. A very strong inhibition was found: when the reconstitution was performed at 0.5 x 10(-6) M viomycin the particles formed sedimented at about 50S, but showed a residual activity of less than 10%. The inhibitory power of viomycin with respect to the in vitro assembly is more pronounced than that found in in vitro systems for protein synthesis.     

Induction of mitochondrial migration in the slime mold Physarum polycephalum

Mitochondrial migration in a microplasmodium of Physarum polycephalumwas studied by litgh and electron microscopy. The mitochondriawere dispersed evenly in the microplasmodium of Physarum polycephalumin shaken cultures but when the microplasmodia were left unshakenin a liquid culture for more than 3 hr, the mitochondria migratedtoward the peripheral area and came into contact with an semi-electrontransparent layer beneath the cell membrane. Once the peripherallocalization of mitochondria was established in unshaken culture,subsequent reversal to the shaken cultures induced a reversion.These results suggest that mitochondrial migration is reversiblyindicated by culture condition. (Received June 19, 1978; )     

Isoaccepting lysine transfer ribonucleic acid species of Pseudomonas aeruginosa.

Pseudomonas aeruginosa tRNA was treated with iodine, CNBr and N-ethylmaleimide, three thionucleotide-specific reagents. Reaction with iodine resulted in extensive loss of acceptor activity by lysine tRNA, glutamic acid tRNA, glutamine tRNA, serine tRNA and tyrosine tRNA. CNBr treatment resulted in high loss of acceptor ability by lysine tRNA, glutamic acid tRNA and glutamine tRNA. Only the acceptor ability of tyrosine tRNA was inhibited up to 66% by N-ethylmaleimide treatment, a reagent specific for 4-thiouridine. By the combined use of benzoylated DEAE-cellulose and DEAE-Sephadex columns, lysine tRNA of Ps. aeruginosa was resolved into two isoaccepting species, a major, tRNA Lys1 and a minor, tRNALys1. Co-chromatography of 14C-labelled tRNALys1 and 3H-labelled tRNALys2 on benzoylated DEAE-cellulose at pH 4.5 gave two distinct, non-superimposable profiles for the two activity peaks, suggesting that they were separate species. The acceptor activity of these two species was inhibited by about 95% by iodine and CNBr. Both the species showed equal response to codons AAA and AAG and also for poly(A) and poly(A1,G1) suggesting that the anticodon of these species was UUU. Chemical modification of these two species by iodine did not inhibit the coding response. The two species of lysine of Ps. aeruginosa are truly redundant in that they are indistinguishable either by chemical modification or by their coding response.     

Antibacterial nitroacridine, Nitroakridin 3582: binding to nucleic acids in vitro and effects on selected cell-free model systems of macromolecular biosynthesis

Nitroakridin 3582 (NA) formed complexes with native deoxyribonucleic acid (DNA) and with transfer ribonucleic acid (tRNA) species from Escherichia coli. Spectrophotometric titrations of NA with these nucleic acids produced numerical results from which nonlinear adsorption isotherms were derived. These curves indicated the existence of more than one class of binding sites on the polymers to which NA was bound by more than one process. The stoichiometry of strong binding of NA to double helical DNA was in agreement with a conventional value (1 ligand molecule per 4.2 component nucleotides) for complete intercalation binding. NA inhibited the DNA-dependent DNA polymerase I and RNA polymerase reactions, the first strongly and the second appreciably. These inhibitions corresponded to the extents to which NA inhibits DNA and RNA biosyntheses in vivo. Evidently, NA interferes with the template function of DNA. The drug also inhibited the polymerization of phenylalanine in a cell-free E. coli ribosome-polyuridylic acid [poly (U)] system. The effect paralleled an inhibition of the poly (U)-directed binding of phenylalanyl tRNA to ribosomes. Ethidium bromide acted similarly. The antimalarial drug, chloroquine, stimulated polyphenylalanine synthesis, apparently as a result of stimulating the poly (U)-directed binding of phenylalanyl tRNA to ribosomes.     

Pairing properties of the methylester of 5-carboxymethyl uridine in the wobble position of yeast tRNA3Arg.

At optimum magnesium concentration (10 mM) both yeast tRNA1Arg and tRNA3Arg are able to bind to poly (A,G) and A-G-A in presence of Escherichia coli robisomes. With A-G-G only tRNA1Arg ginds, wherea tRNA3Arg (anticodon mcm5 U-C-U) is not bound. This result means that the methylcarboxymethyl substituant in position 5 of U prevents its wobble with G.     

Conidiation ofNeurospora crassa in submerged culture without mycelial phase

Summary Conidia ofNeurospora crassa shaken in liquid cultures at 46°C for 15 h and then shifted-down to 25°C germinate directly into conidiophores producing new conidia (macroconidia).     

Influence of moisture and aeration on oxygen uptakes in Warburg respiratory experiments with litter and soil

Summary Oxygen uptakes by litter and soil from a beech forest were determined in a Warburg apparatus, using material at field moisture content, at 60 per cent water-holding capacity (WHC), and at saturation (>100 per cent WHC), in unshaken and shaken flasks. In unshaken flasks, oxygen uptakes by materials from some horizons were markedly dependent upon their moisture content. Oxygen uptakes were similar with unshaken materials at 60 per cent WHC and with shaken water-saturated materials; this latter method of determination is preferred for routinely measuring respiratory activity under aerobic conditions. Control of moisture content of the systems appeared necessary to estimate respiratory responses to added solutions of glucose. Significant responses to added glucose were found with materials from all horizons under conditions of adequate moisture and aeration but responses were much less with some unshaken water-saturated materials.     

Nitrogen and phosphorus removal through laboratory batch cultures of microalga Chlorella vulgaris and cyanobacterium Planktothrix isothrix grown as monoalgal and as co-cultures

Batch cultures of the green microalga Chlorella vulgaris and cyanobacterium Planktothrix isothrix and their corresponding co-cultures were grown in municipal wastewater in order to study their growth as well as the nitrogen (NH₄–N) and phosphorus (PO₄³⁻–P) removal. The cultures were grown under two irradiances of 20 and 60 μmol photons m⁻² s⁻¹ in shaken and unshaken conditions. The co-culture of unshaken Chlorella and Planktothrix showed the greatest growth under both irradiances. The monoalgal Planktotrix cultures showed better growth when unshaken than when shaken, whereas Chlorella cultures grew better when mixed, but only at the higher irradiance. The highest percentage of nitrogen removal (up to 80%) was attained by the unshaken co-cultures of Chlorella and Planktothrix. The amount of nitrogen recycled in the biomass reached up to 85% of that removed. Shaken monoalgal cultures of Chlorella showed phosphorus removal under both irradiances. They completely removed the initial phosphorus concentration (7.47 ± 0.17 mg L⁻¹) within 96 and 48 h under 20 and 60 μmol photons m⁻² s⁻¹, respectively.     

In vitro association of selective +RNA species with 28S RNA of mouse cells

28S RNA prepared either from the poly(A) RNA-depleted fraction of mouse embryo culture cells or from 60S ribosome subunits of adult mouse liver is able to bind selective species of tRNAs in an $\text{in vitro}$ hybridization reaction. The bound tRNA consists predominantly of proline tRNA and, in minor amounts, glycine, alanine, and aspartic acid tRNAs. Quantitative analysis revealed that the hybridization of tRNA may involve a 28S RNA subpopulation, which is present in higher quantity in embryo cells than in adult liver of the mouse.     

70-S ribosomes of Escherichia coli have an additional site for deacylated tRNA binding

Escherichia coli 70-S ribosomes contain a third site for tRNA binding, additional to the A and P sites. This conclusion is based on several findings. Direct measurements showed that in the presence of poly(U), when both A and P sites are occupied by Ac[14C]Phe-tRNAPhe, ribosomes are capable of binding additionally deacylated non-cognate [3H]tRNA. If ribosomes in the preparation are active enough, the total binding of labeled ligands amounted to 2.5 mol/mol ribosomes. In the absence of poly(U), when the A site can not bind, the P site and the 'additional' site can be filled simultaneously with Ac[14C]Phe-tRNAPhe and deacylated [3H]tRNA, or with [3H]tRNA alone; the total binding exceeds in this case 1.5 mol/mol ribosomes. The binding at the 'additional' site is not sensitive to the template. [3H]tRNA bound there is able to exchange rapidly for unlabeled tRNA in solution. Deacylated tRNA is preferred to the aminoacylated one. The binding of AcPhe-tRNAPhe was not observed there at all. The 3'-end adenosine is essential for the affinity. The function of the 'additional' site is not known, but its existence has to be considered when tRNA . ribosome complexes are studied.     

Partial purification and properties of a chromatin bound endonuclease from the marine sponge Geodia cydonium

A chromatin bound endonuclease (Mr:107,000) has been extracted and partially purified from the siliceous sponge Geodia cydonium. Disc gel electrophoresis showed that only one enzyme was present in the partially purified preparation which was able to degrade DNA and poly(A). The enzyme liberates oligonucleotides on incubation with poly(A), which are further degraded to yield the 5'-mononucleotide, which has a pI of 6.5 and a pH optimum of 7.5-8.0. Cations are not required for enzymic activity and EDTA does not inhibit the enzyme. Only iodosobenzoic acid was found to completely inhibit the enzyme. The enzyme hydrolysed poly(A), poly(U), poly(C), DNA, poly[d(A-T)], poly[d(G-C)], but not poly (dA) or poly(G).     

Chemical synthesis and cloning of a poly(arginine)-coding gene fragment designed to aid polypeptide purification

A 43-bp DNA duplex coding for poly(arginine) [poly(arg)] has been synthesised by modified phosphotriester procedures. It has been inserted into the BglII and BamHI restriction sites of a cloned synthetic β-urogastrone (Uro) gene, under the control of the trp promoter. Subsequent induction with 3β-indole acrylic acid produces β-Uro with a C-terminal poly(arg) fusion. The raised isoelectric point of this polypeptide fusion facilitates rapid purification by cation exchange chromatography. The C-terminal poly(arg) tail can be readily removed by treatment with carboxypeptidase B.     

Physical studies of the interaction of a calf thymus helix-destablizing protein with nucleic acids

UP1, a calf thymus protein that destabilizes both DNA and RNA helices, dramatically accelerates the conversion of the inactive conformers of several small RNA molecules to their biologically active forms [Karpel, R. L., Swistel, D. G., Miller, N. S., Geroch, M. E., Lu, C., & Fresco, J. R. (1974) Brookhaven Symp. Biol. 26, 165-174]. Using circular dichroic and spectrophotometric methods, we have studied the interaction of this protein with a variety of synthetic polynucleotides and yeast tRNA3Leu. As judged by perturbations in polynucleotide ellipticity or ultraviolet absorbance, the secondary structures of the single-stranded helices poly(A) and poly(C), as well as the double-stranded helices poly[d(A-T)] and poly(U.U), are largely destroyed upon interaction with UP1 at low ionic strength. This effect can be reversed by an increase in [Na+]: half the UP1-induced perturbation of the poly(A) CD spectrum is removed at 0.05 M Na+. The variation of poly(A) ellipticity and ultraviolet absorbance with [UP1]/[poly(A)]p is used to determine the length of single-stranded polynucleotide chain covered by the protein: 7 +/- 1 residues. A model is presented in which the specificity of UP1 for single strands and their concomitant distortion are a consequence of maximal binding of nucleic acid phosphates to a unique matrix of basic residues on the protein. Analogous to the effect on polynucleotides, UP1-facilitated renaturation of yeast tRNA3Leu follows the partial destruction of the inactive tRNA's secondary structure. At the tRNA absorbance maximum, UP1 effects a hyperchromic change of 10%, representing one-third of the secondary structure of the inactive conformer. This change is also clearly observable as a perturbation of the tRNA's circular dichroism spectrum.     

The use of cross-linking platinum reagents in the study of tRNA and mRNA interaction with ribosomes

The use of some bifunctional Pt(II)-containing cross-linking reagents for investigation of structural organization of ribosomal tRNA- and mRNA-binding centres is demonstrated for various types of [70S ribosome.mRNA-tRNA] complexes. It is shown that treatment of the complexes [70S ribosome.Ac[14C]Phe-tRNA(Phe).poly(U)], [70S ribosome.3'-32pCp-tRNA(Phe).poly(U)] and [70S ribosome.f[35S]Met-tRNA(fMet).AUGU6] with Pt(II)-derivatives results in covalent attachment of tRNA to ribosome. AcPhe-tRNA(Phe) and 3'-pCp-tRNA(Phe) bound at the P site were found to be cross-linked preferentially to 30S subunit. fMet-tRNA(fMet) within the 70S initiation complex is cross-linked to both ribosome subunits approximately in the same extent, which exceeds two-fold the level of the tRNA(Phe) cross-linking. All used tRNA species were cross-linked in the comparable degree both to rRNA and proteins of both subunits in all types of the complexes studied. 32pAUGU6 cross-links exclusively to 30S subunit (to 16S RNA only) within [70S ribosome.32pAUGU6.fMet-tRNA(fMet)] complex. In the absence of fMet-tRNAfMet the level of the cross-linking is 4-fold lower.     

Effect of Shaking on the Growth of Diluted Cultures of Tetrahymena

ABSTRACT. Cultures of Tetrahymena pyriformis, T. thermophila and T. pigmentosa have been studied with regard to growth rates in shaken and unshaken flasks. In the standard medium, a minimum doubling time of 170 min was obtained for T. pyriformis at 28° C in the unshaken cultures. If the depth of the medium was less than 1 cm, the gyratoric shaking increased the doubling time to 340 min. The effect of shaking could be reduced by the addition of dextrane. Cells subjected to shaking were observed in different media and at different growth temperatures. If cultures were inoculated with 10⁴ cell·ml⁻¹ or more, the effect of shaking was absent. However, with inoculates of 10³ or 10² cell·ml⁻¹, the doubling times for T. pyriformis increased to 240 and 275 min, respectively. Periods of 2 min shaking followed by rest for 60 min could not induce an effect.     

Shaking 3T3 cells: further studies on diffusion boundary effects

Confluent quiescent cultures of 3T3 cells can be stimulated to grow—with or without a medium change—simply by shaking (20 mm excursions, 3 per sec). Shaking without medium change, or with low serum concentration in fresh medium (4%), has the greatest effect on thymidine labeling and mitotic index compared with unshaken cultures, but high serum concentration (20%) stimulates both shaken and unshaken cultures equally. Even without medium change, the majority of cells can be stimulated to incorporate thymidine and to divide over 3 days, though initial shaking for 7 hr is sufficient to initiate growth. We conclude that growth in confluent 3T3 cell cultures is limited by a diffusion barrier in the microenvironment, probably affecting uptake of a serum component. The limitation can be overcome by increased fluid velocity as an alternative to raising the serum concentration.Shaking also causes increased cell movement and a change in cell morphology (which may be partly due to alterations in serum caused by shaking). Despite increased movement in the cell sheet, emigration from the border of a wound is diminished by shaking, suggesting that chemotaxis may be involved.To test whether loss of contact between the migrating cells is essential for growth stimulation at the border of a confluent layer, wounds were made in unshaken cultures in the presence of cytochalasin B, which prevents locomotion of cells and maintains the original topography. Nevertheless, the thymidine labeling index was still increased along the wound boundary, and included cells which were near but not on the edge. This supports the view that short range effects on cell growth at discontinuities in cell populations can be due to modification of a diffusion barrier and not simply alteration in cell contacts.