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This report concerns the synthesis of the homopolynucleotides poly(6-methylpurinylic acid), poly(6-methoxypurinylic acid), and poly(6-methylthiopurinylic acid). Starting from the nucleosides the nucleoside 5′-phosphates were prepared enzymatically with carrot phosphotransferase whereas the nucleoside 5′-diphosphates were prepared chemically and were polymerized with polynucleotide phosphorylase from M. luteus. Compared to poly(A) the homopolynucleotides at acidic pH formed no double strand and showed no complex formation with poly(U) at neutral pH values. With respect to the stability of the secondary structure as measured by thermal denaturation and enzymatic digestion the polymers represent intermediates between poly(6-deaminoadenylic acid) and poly(A). In contrast to poly(6-deaminoadenylic acid) none of the polynucleotides stimulated the binding of Lys-tRNA to 70 S ribosomes. more...

Summary The oligonucleotide U-U-| 3 H|U-(nh 2 ) 2 U as a mRNA model was bound covalently to the 16 S RNA via the bifunctional guanosine-specific reagent p-chlorocarboxyphenylglyoxal. Since the ribosomes labeled with the oligonucleotide stimulated the binding of Phe-tRNA, it is concluded, that the probe reacted specifically with a guanosine of the 16 S RNA which forms the mRNA binding site of the 30 S ribosome. The binding of the probe was inhibited by poly(U), but stimulated by the presence of Phe-tRNA. 83 % of the radioactivity was found in the 16 S RNA of the ribosomal subunit. After digesting the 16 S RNA with ribonuclease T1 and separating the oligonucleotides formed by gel electrophoresis, the radioactive labeled material was mainly found in one distinct band. more...

This report concerns the synthesis of poly(5-aminouridylic acid) and of 5-aminouridine-containing trinucleotides. Starting from 5-aminouridine the nucleoside 5′-phosphate was prepared enzymatically with carrot phosphotransferase whereas the nucleoside 5′-diphosphate was prepared chemically and polymerised with polynucleotide phosphorylase. The aminouridine-containing trinucleotides were prepared by known enzymatic procedures. Besides an increase of stability in the secondary structure poly(nh25U) forms a triple-stranded complex with poly(A) and stimulates the poly(Phe) synthesis like poly(U). In contrast to U-nh25U-U, the triplet containing the 3′-terminal aminouridine does not stimulate the binding of Phe-tRNA to 70-S ribosomes. This behaviour is discussed with respect to the influence of a modification on the stacking geometry of a codon and the base pairing scheme between the 5′-nucleotide of the anticodon and the 3′-nucleotide of the codon. more...

α-Cytidylic acid from yeast ribonucleic acid The mixture of mononucleotides obtained from a hydrolysate of yeast RNA has been separated on a Dowex-1 X2 column using formic acid gradient elution. The two fractions, eluted ahead of the 2′-β-cytidylic acid, which have a typical cytidine spectrum have been identified as the 2′- and 3′-α-cytidylic acids. The 2′,3′-cyclic phosphate of α-cytidylic acid is not a substrate for pancreatic ribonuclease nor does it inhibit the reaction, while the dianionic 2′- and 3′-mononucleotides are strong inhibitors. more...

1. 1. Analogues of U-U-U in which every single uridine residue was replaced by either 3-deazauridine, 4-deoxyuridine, or 3-deaza-4-deoxyuridine were tested for their template activities in stimulating the binding of Phe-tRNA to ribosomes. All triplets containing 3-deaza-4-deoxyuridine were inactive as templates, whereas replacements of uridine by 3-deazauridine or 4-deoxyuridine resulted in a loss or decrease of activity depending on the position of the substitution. 2. 2. The binding of 3 H-labelled oligonucleotides to ribosomes was dependent on the presence of the coded tRNA. 1 mole U-U-U or its analogue was bound per mole tRNA Phe yeast . 3. 3. Complex formations between tRNA Phe yeast and various 3 H-labelled oligonucleotides were measured by equilibrium dialysis. Whereas (U) 3 -U and (U) 4 -U showed binding constants of 650 l · mole −1 and 3000 l · mole −1 , respectively, complex formation between other oligonucleotides and tRNA Phe was hardly detectable by the technique employed. 4. 4. The observations are discussed in view of the mechanism of coded aminoacyl-tRNA binding to ribosomes. more...

The practical use of thin-layer chromatography and electrophoresis in the separation and identification of oligonucleotides derived from nuclease hydrolyses of a single transfer ribonucleic acid (tRNAPhe from Escherichia coli B) has been explored, with particular attention to limitations of oligonucleotide size and to subsequent recovery and degradation analysis at the 1–10 nanomole level. In this range, where thin-layer methods are most efficient, satisfactory recoveries from the adsorbent are limited to penta- and smaller oligonucleotides, and the subsequent analyses require methods of corresponding sensitivity. The latter need is satisfied by the “nucleoside analyzer” of U ziel , K oh and C ohn . The most efficient use of thin-layer methods appears to be separation, in one dimension, of mixed-peaks derived from a prior column separation of the original enzymic digest of the tRNA in question. more...

The impurity-induced nucleation and the kinetics of the growth of filamentary crystals of silicon and germanium from silane and germane respectively have been studied in a closed system. Silicon whiskers were grown on heated substrates at temperatures between 550 and 900 °C, germanium whiskers at temperatures between 300 and 500 °C, both at hydride pressures between 5 and 100 Torr, and with Au as impurity. Whisker growth was also obtained with Ag, Cu, Ni and Pd, but not with In, Sn and Bi. The rates of growth of the whiskers in the length direction increase with increasing substrate temperature and hydride pressure, and are of the order of a hundred times higher than the rates of thickness growth of whiskers and substrate. As compared with the non-impurity aided deposition of Si and Ge from the hydrides, where there is an exponential relation between deposition rate and reciprocal temperature, the effect of the growth stimulus at the tip is reflected in a decrease of the activation energy and an increase in the pre-exponential factor. In terms of the VLS mechanism the catalyzed decomposition reaction at the V-L interface is the rate-controlling step. more...

1. Analogues of U-U-U in which uridine residues were replaced by either 2-thiouridine, 4-thiouridine, 2,4-dithiouridine, or 2-thiocytidine were synthesized by enzymatic methods. 2. The dinucleoside phosphates U-N and the trinucleoside diphosphates U-U-N were prepared by ribonuclease A-catalyzed transesterification. 3. The dinucleoside U-N was elongated by using primer-dependent polynucleotide phosphorylase to yield U-N-U. 4. For the synthesis of N-U-U the dinucleoside phosphate G-N, prepared with ribonuclease T 1 , was used as a primer in the polynucleotide phosphorylase-catalyzed reaction to yield G-N-U-U, from which the initial Gp was split off by ribonuclease T 1 . 5. The coding properties of the newly synthesized trinucleoside diphosphates are reported. It follows that Ado·s 2 Urd forms a strong base pair, whereas the base pairs Guo·s 4 Urd, Guo·s 2 Cyd, and Ado·s 4 Urd are weak. No interaction between guanosine and 2-thiouridine or 2,4-dithiouridine could be detected in the binding assay. more...

THE nucleoside analogue 3-deazauridine1,2 (Fig. 1), 4-hydroxy-1-(β-D-ribo-pentafuranosyl)-2-pyridone, has powerful cytostatic properties; at a concentration of 5 × 10−6 M it inhibits the growth of Ehrlich ascites cells by 50% (ref. 3). In the codon-specific binding of tRNAphe to ribosomes, 3-deazauridine can substitute for uridine in the 5′ - and middle position of the codon but not in the wobble position4. NMR evidence1,2 suggests that the major form in solution has a 4-hydroxyl group but a dynamic enol-keto tautomerism is taking place. The present X-ray diffraction study was undertaken to determine the tautomeric form present in the crystalline state and to investigate the hydrogen bonding possibilities of 3-deazauridine. We find the enol form with an unusually short hydrogen bond between the 4-hydroxyl group and the 2-keto oxygen of a neighbouring molecule. more...

A model is proposed which assumes that the codon-anti-codon interaction induces a conformational change in the tertiary structure of AA-tRNA exposing the hidden T-Ψ-C-G sequence for binding to the C-G-A-A sequence in the 5S RNA of the 50S ribosomal subunit. The model is supported by binding experiments with C-G-(3H)A-(3H)A and by the inhibition of poly(Phe) synthesis by C-G-A-A. The process is dependent on the 30S subunit, the template, and the AA-tRNA × EF-Tu × GTP complex. more...

Utilização de serviçp de saúde; Sistema Único de Saúde (SUS); Programa de Saúde da Família (PSF).

The homopolynucleotides poly(c1A), poly(c3A), poly(c7A) and poly(h6A) ++ were synthesized from their corresponding nucleoside diphosphates using polynucleotide phosphorylase. With the exception of poly(h6A), which displayed no hypochromicity, the homopolynucleotides showed melting profiles similar to poly(A). All these polynucleotides, poly(h6A), poly(c7A), poly(c3A) and poly(c1A) stimulated the binding of Lys-tRNA to ribosomes; the coding activity of poly(c1A), however, was very low. Poly(h6A) was found to be less specific for Lys-tRNA than poly(A). The data supports the exclusive formation of Watson-Crick type base pairs and contradicts Hoogsteen base pairing in codon-anticodon recognition. Since, however, poly(h6A), which can form only one hydrogen bridge per base pair, stimulated the binding of Lys-tRNA comparably to poly(A), the coding activity of the homopolynucleotides tested is discussed in respect to their secondary structure as well as to the pK-values of their 6-amino groups. more...

Summary Polymer-bound ribonuclease has been used to synthesize the termination codons U-A-A, U-A-G, and U-G-A. RNase bound to carboxymethyl-cellulose shows no bleeding and can be re-used without loss of activity. The method was used with 1 μmole and 50 μmoles of starting materials. The yields are 4–18% depending on the solubility of the dinucleoside phosphate. All materials are commercially available. The main advantage of this synthetic method lies in the simple and fast procedure. more...