In vitro incorporation of 2′-deoxyadenosine and 3′-deoxyadenosine into yeast tRNAPhe using tRNA nucleotidyl transferase,and properties of tRNAPhe-C-C-2′dA and tRNAPhe-C-C-3′dA

2′-Deoxyadenosine and 3′-deoxyadenosine (cordycepin) can be incorporated into the 3′-terminal position of tRNA^Phe by tRNA nucleotidyl transferase. tRNA^Phe-C-C-2′dA and tRNA^Phe-C-C-3′dA, missing the cis-diol group at the 3′-terminal end are resistant to periodate oxidation and are not able to form borate complexes. In aminoacylation experiments only the tRNA^Phe-C-C-3′dA proved to be chargeable.     

Determination of the degree of in vivo tRNA aminoacylation in yeast cells

A method is described to harvest yeast cells, extract tRNAs having a good biological activity, and measure the degree of in vivo tRNA aminoacylation. This measuremet is based on a determination of the percentage of tRNA which is resistant to periodate oxidation; control experiments have been performed to check that this treatment inactivates uncharged tRNA molecules, but does not affect aminoacylated ones, and allows therefore an accurate determination of the percentage of aminoacylated tRNA molecules.     

Periodate oxidation studies in the elucidation of the structures of sialic acid containing oligosaccharides

The structures of sialo-oligosaccharide alditols as determined by ¹H-NMR spectrometry together with methylation analysis did not correspond with those derived previously from quantitative periodate oxidation data alone. Possible causes of the discrepancy were explored in the periodate oxidation methodology. No free sialic acid was released by the acidity of the periodate in the course of oxidation at pH 4.5. The anionic properties of the sialic acid residues were therefore utilized to separate the periodate oxidation products and thereby establish the position of the sialic acid in the oligosaccharide chain.     

In Vivo Aminoacylation of Transfer Ribonucleic Acid in Bacillus subtilis and Evidence for Differential Utilization of Lysine-Isoaccepting Transfer Ribonucleic Acid Species

The presence or absence of certain amino acids has different effects on the ability of Bacillus subtilis to sporulate, and the intracellular pool size of amino acids has been reported to vary during sporulation. The idea that these variations might exert a regulatory effect through aminoacylation of transfer ribonucleic acid (tRNA) was investigated by studying the levels of aminoacylation in vivo in the logarithmic or stationary phase of growth. Both the periodate oxidation method and the amino acid analyzer were used to evaluate in vivo aminoacylation. The results indicated that in general the level of aminoacylation of tRNA's remained constant through stage III of sporulation, although there were detectable variations for specific amino acid groups. Our studies also showed that periodate oxidation damaged certain tRNA's; therefore, the results obtained by such a method should be interpreted with caution. Because the damage can affect certain isoaccepting species specifically, the periodate oxidation method cannot be used to establish which isoaccepting species are acylated in vivo. We also investigated the possibility of preferential use of particular tRNA species by polyribosomes. These results demonstrated a preferential use of lysyl-tRNA's at different growth stages. Control mechanisms operating during the early stages of sporulation, therefore, do not affect the overall level of aminoacylation. However, there is an effect on the levels of aminoacylation of specific amino acids and on which isoaccepting species are utilized by the polyribosome system.     

The role of modified purine 64 in initiator/elongator discrimination of tRNA(iMet) from yeast and wheat germ.

The role of 2'-ribosylated adenosine 64 in tRNA(iMet) from yeast in initiation/elongation discrimination was investigated. As measured by in vitro translation in rabbit reticulocyte lysate, the specific removal of the 2'-ribosylphosphate at adenosine 64 via periodate oxidation allows tRNA(iMet) to read internal AUG codons of the globine messenger RNA. Yeast Met-tRNA(iMet) lacking the modification of nucleoside 64 forms ternary complexes with GTP and elongation factor Tu from Escherichia coli. The lack of modification at position 64 does not prevent tRNA(iMet) from participating in the initiation process of in vitro protein synthesis. Wheat germ tRNA(iMet) has a 2'-ribosylated guanosine at position 64. Removal of this modification from the wheat germ tRNA(iMet) enables it to read internal AUG codons of globine and tobacco mosaic virus messenger RNA in reticulocyte and wheat germ translation systems, respectively.     

Aminoacylation of four tRNA species in lupin (Lupinus luteus) cotyledons

During germination of lupin seeds, the levels of in-vivo tRNA aminoacylation increase in different ways, depending on the species of tRNA. Column chromatography of tRNA on reverse-phase-chromatography (RPC-5) has shown the presence of 4 peaks of isoleucyl-tRNA, 5 of leucyl-tRNA, 5 of lysyl-tRNA, 2 of tyrosyl-tRNA, and 4 of valyl-tRNA. Cochromatography of periodate treated and control tRNA preparations, labeled with radioactive amino acids, indicates identical aminoacylation in vivo of isoaccepting tRNAs during plant development. One isoacceptor of isoleucine tRNA changes its elution profile after periodate treatment.Abbreviation RPC-5 reverse-phase-chromatography     

Discrimination between initiation and elongation of protein biosynthesis in yeast: identity assured by a nucleotide modification in the initiator tRNA.

Cytoplasmic initiator tRNAs from plants and fungi possess an unique 2'-phosphoribosyl residue at position 64 of their sequence. In yeast tRNA(iMet), this modified nucleotide located in the T-stem of the tRNA is a 2'-1'-(beta-O-ribofuranosyl-5'-phosphoryl)-adenosine. The phosphoribosyl residue of this modified nucleoside was removed chemically by treatment involving periodate oxidation of tRNA(iMet) and regeneration of the 3'-terminal adenosine with ATP (CTP):tRNA nucleotidyl transferase. The role of phosphoribosylation at position 64 for interaction with elongation factor eEF-1 alpha and initiation factor 2 (eIF-2) was investigated in the homologous yeast system. Whereas the 5'-phosphoribosyl residue prevents the binding of Met-tRNA(iMet) to eEF-1 alpha, it does not influence the interaction with eIF-2. After removal of the ribosyl group, the demodified initiator tRNA showed binding to eEF-1 alpha, but no change was detected with respect to the interaction with the initiation factor eIF-2. This observation is interpreted to mean that a single modification of an eucaryotic initiator tRNA in yeast serves as a negative discriminant for eEF-1 alpha, thus preventing the initiator tRNA(iMet) from entering the elongation cycle of protein biosynthesis.     

Plasmodium berghei: modification of sialic acid on red cells from infected mouse blood

The surface membrane glycoproteins of normal mouse erythrocytes can be labeled by oxidation with either periodate or galactose oxidase in the presence of neuraminidase, followed by reduction with NaB³H₄. Without neuraminidase there is little galactose oxidase-catalyzed labeling of protein. Analysis of labeled proteins by SDS-polyacrylamide gel electrophoresis showed that both methods labeled the same set of glycoproteins. Plasmodium berghei infection dramatically reduced the sialoglycoprotein labeling of red blood cells from infected blood using the periodate/NaB³H₄ method. Provided neuraminidase was present, labeling by the galactose oxidase method gave identical results to normal erythrocytes. We conclude that the glycoprotein sialic acid of uninfected as well as infected red cells is modified during infection such that it is refractory to periodate oxidation. Acylation of the exocyclic hydroxyls of sialic acid is suggested to account for this. Lectin binding and cell agglutination experiments using Limulin, soybean and wheatgerm lectins, and concanavalin A confirmed and extended these observations. The possible implications of these results with regard to anemia induced by malaria are briefly discussed.     

Status of tRNA charging, trinucleotide acceptor sequence and tRNA nucleotidyltransferase activity in the human placenta.

Samples of tRNA isolated from the cell sap of full-term human placenta were found to have a low capacity for accepting amino acids in the presence of partially purified synthetase preparations made from placental or rat liver cell sap. Gel electrophoresis of placental tRNA showed that part of this could be accounted for by gross degradation. The proportion of chargeable tRNA carrying amino acids was estimated by periodate oxidation followed by stripping and then charging with labeled amino acids. Only 50% of chargeable placental tRNA was in the charged state when isolated, whereas 87% of freshly isolated rat liver tRNA was found to be charged with amino acids. A fraction from placental cell sap was shown to have tRNA nucleotidyltransferase activity. When placental tRNA was incubated with this fraction and [3H]ATP or [3H]CTP, ATP was incorporated into about 12% of the tRNA molecules and CTP into 5-7%. When rat liver tRNA was used in place of placental tRNA, [3H]ATP was incorporated into less than 5% of the tRNA molecules. By using snake-venom diesterase over short periods of incubation, it was confirmed that the ATP had been incorporated terminally as AMP into the placental tRNA. These observations show that, in contrast to rat liver tRNA, tRNA prepared from human placenta is poorly charged with amino acids, many of the molecules lack the acceptor trinucleotide and there is extensive degradation beyond this stage.     

Immobilization of lipase from Candida rugosa on Sepabeads(®): the effect of lipase oxidation by periodates

The objective of this paper was the investigation of a suitable Sepabeads^? support and method for immobilization of lipase from Candida rugosa. Three different supports were used, two with amino groups, (Sepabeads^? EC-EA and Sepabeads^? EC-HA), differing in spacer length (two and six carbons, respectively) and one with epoxy group (Sepabeads^? EC-EP). Lipase immobilization was carried out by two conventional methods (via epoxy groups and via glutaraldehyde), and with periodate method for modification of lipase. The results of activity assays showed that lipase retained 94.8% or 87.6% of activity after immobilization via epoxy groups or with periodate method, respectively, while glutaraldehyde method was inferior with only 12.7% of retention. The immobilization of lipase, previously modified by periodate oxidation, via amino groups has proven to be more efficient than direct immobilization of lipase via epoxy groups. In such a way immobilized enzyme exhibited higher activity at high reaction temperatures and higher thermal stability.     

Control of Isoleucine, Valine, and Leucine Biosynthesis VI. Effect of 5′,5′,5′-Trifluoroleucine on Repression in Salmonella typhimurium

The leucine analogue 5',5',5',-trifluoroleucine (fluoroleucine) replaced leucine for repression of the isoleucine-valine biosynthetic enzymes in Salmonella typhimurium. In contrast, the analogue had no effect on derepression of the leucine biosynthetic enzymes in leucine auxotrophs grown on limiting amounts of leucine. The effect of fluoroleucine on repression appeared to be specific for leucine since derepression of the isoleucine-valine enzymes due to an isoleucine or valine limitation was not affected by the analogue. The prevention of derepression by fluoroleucine was probably due to repression and not to the formation of false proteins, since the analogue had no effect on the derepression of a number of enzymes unrelated to the isoleucine-valine pathway. Fluoroleucine was able to attach to leucine transfer ribonucleic acid (tRNA) as evidenced by the ability of the analogue to protect about 70% of leucine tRNA from oxidation by periodate. We propose that the differential effects of fluoroleucine on repression are due to differences in the ability of the analogue to bind to the various species of leucine tRNA.     

Prevention of overoxidation in periodate oxidation of reducing oligosaccharides by their conversion into 1,5-anhydroalditol derivatives

A procedure for the conversion of reducing oligosaccharides into their 1,5-anhydroalditol derivatives was devised to prevent overoxidation during periodate oxidation. Gas-chromatographic analysis of the aldehydes in the products of complete oxidation of the resultant 1,5-anhydroalditol derivatives by the dithioacetal method^1b indicated that new types of dialdehyde characteristic of the linkage-types were formed, together with ordinary simple aldehydes. A number of d-gluco-oligosaccharides having various types of interglycosidic linkage were examined by this method. The results were consistent with expectations.     

The application of PEI-cellulose thin-layer chromatography for the resolution of large oligonucleotide fragments of transfer ribonucleic acids.

Large oligonucleotide fragments from tRNA were separated on PEI-cellulose tle using stepwise gradients of increased concentrations of LiCl (containing 0.3 m Tris-HCl and 7.5 m urea at pH 7.9) or Li-formate (containing 7.5 m urea at pH 3.5). These large oligonucleotides, obtained by cleavage of tRNA with nuclease S₁, aniline-NaOH, or partial ribonuclease T₁ digestion and separated on PEI-cellulose, were analyzed by three different methods. The first method entailed elution and total base analysis by the tritium-postlabeling technique; the second involved complete ribonuclease T₁ digestion in situ, contact transfer to another PEI-cellulose tle plate, and two-dimensional tle fingerprinting; the third employed complete digestion in situ with ribonuclease T₁ and bacterial alkaline phosphatase, followed by the elution, periodate oxidation, introduction of a tritium into 3′-terminus, and subsequent two-dimensional PEI-cellulose fingerprinting. These techniques can aid in the determination of the complete nucleotide sequence of tRNA when only small quantities of pure tRNAs (less than 10 A₂₆₀ units) are available or when the tRNAs are not amenable to in vivo radioactive labeling.     

The kinetics of suppression of proliferation of oxidized murine lymphoid cells by sodium borohydride reduction.

Murine splenic lymphoid cells are stimulated to proliferate following mild oxidation with sodium periodate. To assess the class of cells responding, we used periodate treatment alone or in association with concanavalin A, a T-cell mitogen, or lipopolysaccharide (LPS), primarily a B-cell mitogen. Brief periodate treatment followed by culturing with concanavalin A gave no additive proliferative response to that seen using concanavalin A alone, while culturing periodate-treated cells with LPS gave approximately an additive response. Furthermore, periodate failed to stimulate spleen cells from neonatally thymectomized mice while LPS produced significant stimulation of proliferation, suggesting that periodate is stimulating a class of T lymphoid cells or a subpopulation of T cells. Studies were performed to determine an optimal concentration of borohydride which would suppress proliferation in lymphoid cells initially oxidized with periodate. It was observed that 2 mM borohydride would suppress proliferation of oxidized cells yet permit a normal response of these cells to another T-cell mitogen, concanavalin A. Higher concentrations of borohydride, from 3 to 5 mM, would also suppress proliferation of oxidized cells but would interfere with the ability of these cells to respond to concanavalin A, perhaps due to cell damage. Studies were performed to determine when it was possible to suppress periodate-induced mitogenesis by reducing with borohydride at various times after the initial oxidation. It was observed that 2 mM borohydride treatment could suppress stimulation through 8 hr after the original periodate oxidation and that from 12 hr through 20 hr after the initial periodate oxidation, borohydride was incapable of inhibiting proliferation. Additional studies demonstrate that optimal mitogenesis induced by periodate or concanavalin A is contingent upon a serum factor.     

Periodate oxidation of polysaccharides for modification of chemical and physical properties

A limited degree (typically 1-20%) of periodate oxidation of polysaccharides may give rise to derivatives with entirely altered chemical and physical properties. Notably, the ring opening caused by periodate leads to the formation of highly flexible ‘hinges’ in otherwise rather semiflexible or rigid structures. This review highlights selected examples with the main focus on periodate oxidation of alginates, chitosans, hyaluronan, scleroglucan/schizophyllan, and cellulose.     

O-ribosyl-phosphate purine as a constant modified nucleotide located at position 64 in cytoplasmic initiator tRNAs(Met) of yeasts.

The unknown modified nucleotide G*, isolated from both Schizosaccharomyces pombe and Torulopsis utilis initiator tRNAs(Met), has been identified as an O-ribosyl-(1"----2')-guanosine-5"-phosphate, called Gr(p), by means of HPLC, UV-absorption, mass spectrometry and periodate oxidation procedures. By comparison with the previously published structure of Ar(p) isolated from Saccharomyces cerevisiae initiator tRNA(Met), the (1"----2')-glycosidic bond in Gr(p) has been postulated to have a beta-spatial conformation. The modified nucleotide Gr(p) is located at position 64 in the tRNA(Met) molecules, i.e. at the same position as Ar(p). Since we have also characterized Gr(p) in Candida albicans initiator tRNA(Met), the phosphoribosylation of purine 64 can be considered as a constant nucleotide modification in the cytoplasmic initiator tRNAs(Met) of all yeast species so far sequenced. Precise evidence for the presence of Gr(p) in initiator tRNAs(Met) of several plants is also reported.     

A two-dimensional thin layer chromatographic procedure for the sequential analysis of oligonucleotides employing tritium post-labeling.

Two dimensional PEI-cellulose thin layer chromatography can resolve sequentially degraded oligonucleotide fragments of tRNA. This technique entails the sequential degradation of the oligonucleotide with snake venom phosphodiesterase in the presence of bacterial alkaline phosphatase, and periodate oxidation followed by tritiated sodium borohydride reduction of the 3' terminal nucleoside. Subsequently the tritiated oligonucleotide fragments were resolved by two dimensional PEI-cellulose TLC. The results of these experiments indicate that, in some cases, the complete nucleotide sequence of a large oligonucleotide fragment may be determined by interpretation of the observed mobility shifts, thereby eliminiating the need for additional analysis of the oligonucleotide. In addition, the use of two-dimensional rather than one-dimensional resolution of the tritium labeled fragments allows for a complete separation of any interfering background spots from the sequentially degraded oligonucleotides. This procedure was applied to the complete nucleotide sequence analysis of several ribonuclease T1Val and ribonuclease A digestion products from human placenta tRNA.     

Inhibition by sodium periodate of the in vitro antibody response of mouse spleen cells and its reversal by borohydride or aldehyde blocking reagents

Mild oxidation of mouse spleen cells by sodium periodate induces blastogenesis with enhancement of thymidine incorporation. Concomitantly, the specific in vitro response of these cells to sheep red blood cells and trinitrophenyl-polyacrylamide and the nonspecific polyclonal B-cell response to lipopolysaccharide are markedly inhibited. Exposure of these cells to sodium borohydride and hydroxylamine following periodate treatment reduces blastogenesis and prevents periodate-induced suppression. Data suggest that the integrity of aldehyde moieties generated by periodate oxidation is necessary for blastogenesis and induction of suppressor cells in mouse spleen cell culture.     

Synthesis of novel water-soluble sulfonated cellulose

Water-soluble sulfonated cellulose (SC) samples were synthesized by oxidizing hardwood kraft pulp with sodium periodate followed by the sulfonation reaction with sodium bisulfite. Six levels of oxidation/sulfonation were obtained by using different mmols (0.93-4.67) of periodate per gram of pulp. The aldehyde and sulfonic acid contents, surface morphology, and water solubility property of these treated fibers were characterized. It was found that carbonyl group content increased with the periodate charge and so did the sulfonic acid content in subsequent sulfonation step. Scanning electron microscopy images showed a significant change in surface morphology of the sulfonated samples. Solubility of sulfonated cellulose in water was determined from ¹H NMR spectra and a solubility of 28.57 g/L was found when cellulose was oxidized with 4.67 mmol periodate per gram cellulose followed by the sulfonation reaction.