Interspecies transfer by immune RNA of lymphocyte proliferative response to specific antigen

Ribonucleic acid (RNA) extracted from the lymph nodes of BCG sensitized cattle transferred tuberculin sensitivity to normal guinea pig lymphocytes as indicated by increased incorporation in vitro of ³H-thymidine in response to Purified Protein Derivative (PPD). The RNA treated lymphocytes were unresponsive to a nonspecific antigen, histoplasmin. Ribonuclease treatment of the RNA abolished its ability to transfer tuberculin reactivity and RNA extracted from the lymph nodes of normal cattle was also ineffective.     

Mechanism of codon recognition by transfer RNA and codon-induced tRNA association

The steps of UUC recognition by tRNAPhe were analysed by temperature-jump measurements. At ion concentrations close to physiological conditions we found three relaxation processes, which we assigned to (1) formation of codon-anticodon complexes, (2) a conformational change of the anticodon loop coupled with Mg2+ binding, and (3) codon-induced association of tRNA. The relaxation data were evaluated both by the usual procedure (fitting the exponentials evaluated from the individual experiments of a set to a reaction model) and by "global fitting", i.e. fitting a set of relaxation curves obtained at various concentrations directly to a reaction model, thus leaving out the intermediate exponential fitting step. The data can be represented quantitatively by a three-step model: the codon binds to the anticodon at a rate of 4 X 10(6) to 6 X 10(6) M-1S-1 as is usual for the formation of oligomer helices; the conformation change of the anticodon loop is associated with inner sphere complexation of Mg2+ at a rate of 10(3) S-1; the codon-tRNA complexes form dimers at a rate of 5 X 10(6) to 15 X 10(6) M-1S-1. A similar mechanism is found for the binding of the wobble codon UUU to tRNAPhe at increased concentrations of Mg2+. Measurements at different Mg2+ concentrations demonstrate the distinct role of this ion in the codon recognition and the codon-induced tRNA dimerization. We propose a simple mechanism, based upon the special properties of magnesium ions, for long-distance transfer of reaction signals along nucleic acid chains.     

Release of the inhibition of messenger RNA translation in extracts of interferon-treated Ehrlich ascites tumor cells by added transfer RNA

In an extract of Ehrlich ascites tumor (EAT) cells which had been “preincubated” for 45 min to lower endogenous protein synthesis (S30_C) the translation of exogenous encephalomyocarditis (EMC) viral mRNA proceeds at a constant rate for over 90 min. In a similarly treated extract of interferon-treated EAT cells (S30_INT) the translation proceeds at a lower rate than in the S30_C for about 30 min and then stops. The impairment of the translation in the S30_INT is mediated by one or more inhibitors. After the cessation of translation the viral mRNA in the S30_INT is in large polysomes. The size of these changes little (if any) during a further 15 min incubation. The addition of mouse tRNA (but not ribosomal RNA or $\text{E. coli}$ tRNA) to the S30_INT after the cessation of viral mRNA translation results in the restart of translation at a rate close to that in the S30_C. This effect of tRNA is diminished by pactamycin, which inhibits peptide chain initiation but not elongation. These results indicate that addition of tRNA allows the elongation of incomplete peptide chains and the initiation of new chains. The need for added tRNA may be due to the fact that in S30_INT the amino acid acceptance of some of the endogenous tRNA species (but not of added tRNAs) is impaired. This impairment is pronounced for leucine and very slight, if any, for five other amino acids tested (i.e. isoleucine, methionine, phenylalanine, threonine, and valine).     

Mechanism of codon recognition by transfer RNA studied with oligonucleotides larger than triplets.

The binding of yeast tRNAPhe to UUCA, UUCC, UUCCC, UUCUUCU, U4, U5, U6 and U7 was analysed by fluorescence temperature jump and equilibrium sedimentation measurements. In all cases the two observed relaxation processes can be assigned to alpha) an intramolecular conformation change of the anticodon loop and beta) preferential binding of the oligonucleotides to one of the anticodon conformations. The anticodon loop transition is associated with inner sphere complexation of Mg2+ and proceeds with rate constants of about 10(3) s-1. The rate constants of oligonucleotide binding are between 4 and 10 X 10(6) M-1s-1 and reflect an increase of the association rate with the number of binding sites compensated to some degree by electrostatic repulsion in the preequilibrium complex. Neither temperature jump nor equilibrium sedimentation experiments provided evidence for UUCA or UUCC induced tRNA dimerisation, although UUC binding leads to strong tRNA dimerisation under equivalent conditions. The results obtained for the longer oligonucleotides are similar. In the case of UUCUUCU with its two potential binding sites for tRNAPhe there was no evidence for the formation of 'ternary' complexes. Apparently tRNAPhe binds preferentially to the second UUC of this 'messenger' and forms additional contacts with residues on either side of the codon. Some evidence for the formation of ternary complexes is obtained for U6 and U7, although the extent of this reaction remains very small. Our results demonstrate that the mode of tRNA binding to a codon is strongly influenced by residues next to the codon. The formation of cooperative contacts between tRNA molecules at adjacent codons apparently requires support by a catalyst adjusting an appropriate conformation of messenger and tRNA molecules.     

Mechanism of suppression in Drosophila: a change in tyrosine transfer RNA

The mechanism of suppression of the vermilion locus in Drosophila melanogaster is examined. The suppressor locus, su(s)², is shown to control directly the amount of a specific tyrosine transfer RNA which occurs in the adult fly. Wild-type flies have three chromatographic forms of tyrosine tRNA but flies that are homozygous for the suppressor gene su(s)² contain little or none of the second chromatographic form. The isoacceptor patterns of tRNA for leucine, phenylalanine and serine are identical in the suppressor mutant and wild-type fly. Genetic data show that the phenotypic expression of su(s)² and the altered chromatographic pattern of tyrosine tRNA are recessive and that both map at the same position on the left tip of the X chromosome. Furthermore, another suppressor of vermilion was induced by ethyl methane sulfate, su(s)^e1, that is at the same locus as su(s)² and that produces the same change in tyrosine tRNA as su(s)².     

Alkylation of transfer RNA by N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea

The alkylation of a number of purified tRNA preparations by reaction with the carcinogens, N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea was studied in order to investigate the role of nucleic acid structure on the distribution of alkylation products within the nucleotide sequence. The rate of alkylation was greatly increased by increasing the pH over the range 6 to 8 and the degree of alkylation (expressed as moles alkyl groups/mole tRNA) was directly proportional to the concentration of the nitrosamide added and independent of the amount of tRNA present. There was no significant difference in the degree of alkylation of any of the tRNA preparations tested. Reaction with N-ethyl-N-nitrosourea resulted in a degree of alkylation some 13 times less than that produced by reaction with a similar concentration of N-methyl-N-nitrosourea. The major product of the reaction was 7-alkylguanine amounting to about 80% of the total, but 3-methylcytosine, 6-O-methylguanine and 1-methyl-, 3-methyl-, and 7-methyladenine were also identified as products of the reaction of tRNA^fMet with N-methyl-N-nitrosourea.The possibility that preferential alkylation of certain residues within the polynucleotide sequence was produced by reaction with the nitrosamides was examined by degradation of the alkylated tRNA with pancreatic ribonuclease and separation of the oligonucleotide fragments by chromatography on DEAE cellulose. When tRNA^fMet which had been alkylated by reaction with N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea was analysed in this way, the distribution of 7-alkylguanine was, within the limits of experimental error, in agreement with that expected for a random reaction of the alkylating agent with all of the guanosine residues throughout the molecule. A similar result was seen when tRNA^Phe was examined. These results were obtained by alkylation under conditions where the native configuration of the tRNA was maintained and show that the tertiary structure of the nucleic acid does not impart any specificity to the reaction with the nitrosamide producing 7-alkylguanine but the possibility that such specificity does exist for the minor products of alkylation cannot be excluded.     

Hydrolysis of RNA monomers by extracts of Aspergillus niger NRRL3

Extracts of Aspergillus niger NRRL₃ catalyzed dephosphorylation of AMP, GMP, CMP and UMP over a wide range of pH values from pH 1.5 to pH 10. They also catalyzed hydrolytic deamination of only cytidine out of the tested ribonucleotides, ribonucleosides and bases. Neither cleavage of the N-glycosidic linkages of these nucleotides nor those of the corresponding nucleosides could be effected by the extracts. Phosphate liberation from the four RNA monomers seemed to be effected by two phosphate-non repressible phosphatases, acid and alkaline. Optimum activity of the acid phosphatase with all the substrates was at pH2 and 40 °C while that of the alkaline phosphatase was at pH8 and 40 °– 70 °C. Affinities of both phosphatases for the different ribonucleotides were in the order of magnitude AMP, CMP and phph > GMP > UMP. Freezing and thawing of the extracts had no effect either on the activities of two phosphatases or on that of the aminohydrolase. However, heating the extracts at 55° for 25 min, in absence of the substrate, inactivated the phosphatases and had no effect on the deaminase. No evidence for the involvement of specific nucleotidases in ribonucleotides dephosphorylation was recorded.