Hubert Chantrenne

The Search for the Messenger RNA of Hemoglobin

Publisher Summary This chapter describes the search for the messenger RNA (mRNA) of hemoglobin. Synthetic polynucleotides can certainly fulfill the main function of natural mRNA. A viral RNA is a homogeneous molecular species that contains the information for several proteins, three in the simplest viruses and in some defective viruses perhaps only one. Reticulocyte RNA was centrifuged on a sucrose gradient and separated into four fractions that were assayed for stimulatory activity. The fraction containing 23 S ribosomal RNA was slightly inhibitory, those containing either 16 S or 4S RNA gave a slight stimulation, but fraction 111 that contains a part of the light side of the 16s peak and the region of the gradient between 16s and 4S, did stimulate hemoglobin synthesis in the cell-free reticulocyte system. The fact that the polypeptides made in vitro correspond to hemoglobin, shows that the system is correctly phased. A rough estimate of the amount of mRNA in reticulocyte polyribosomes as compared with ribosomal RNA (rRNA) indicated that it should be possible to detect its ultraviolet light absorption, by merely scaling up the operations, by a factor of ten.

Degradation of deadenylated rabbit α globin mRNA in Xenopus oocytes is associated with its translation

THE 3′-OH poly(A) segment of globin mRNA ensures the stability of the message when injected into Xenopus oocytes1–4. We recently developed5 a specific method for the removal of the poly(A) tail from globin mRNA, based on synchronous processive phosphorolysis of mRNA using molar excess of E. coli polynucleotide phosphorylase at 0 °C. When injected into Xenopus oocytes, deadenylated globin mRNA is translated for a relatively short period and then rapidly degraded1,2. Native poly(A)-containing mRNA, however, is considerably more stable in the same conditions and is translated for extended periods of time1,2,7. The poly (A) segment itself is responsible for the stability of native globin mRNA in Xenopus oocytes since poly(A) re-addition to previously deadenylated mRNA restores the functional stability of the message3. We do not yet know, however, how poly(A) exerts its protective function; furthermore, the mechanism of degradation of poly(A)-free mRNA is not known. We report here the use of the enhancing effects of haemin on the translation of α-globin mRNA in frog oocytes6 to establish that, in these cells, the degradation of injected poly(A)-free α-globin mRNA is linked to its translation.

The binding of proflavine to transfer ribonucleic acid: Dependence on secondary structure

Abstract Proflavine (3,6-diaminoacridine) binds strongly to transfer RNA (tRNA) from Escherichia coli . Direct evidence is obtained by ultracentrifugation, gel electrophoresis and molecular filtration of the complex. Studies on the stability of the tRNA-proflavine complex as a function of temperature show that strong binding depends on the secondary structure of the nucleic acid. Proflavine stabilizes the macromolecule against thermal denaturation. The results are comparable to those obtained with proflavine and DNA. The complexes of proflavine with tRNA and with DNA are probably of a similar nature.

The relationship of ribonucleic acid to the in vitro incorporation of radioactive glycine into the proteins of reticulocytes

Abstract 1. 1. The rate of incorporation of 14 C-glycine into the proteins of reticulocytes and their RNA content have been determined as a function of the development and disappearance of a phenyl-hydrazine induced reticulocytosis in rabbits. 2. 2. The peak of labelled amino acid incorporation occurred 1 to 2 days before the RNA peak. 3. 3. The formation of carbonic anhydrase, hemoglobin and peptidase corresponds quite closely with the RNA curve rather than with the 14 C glycine incorporation curve. 4. 4. These results are interpreted to indicate that RNA is not involved in the initial incorporation of amino acids into proteins but rather mediates the differentiation of a “primary” protein into its final stereospecific, biologically active form.

Action de la 8-azaguanine sur la synthèse des protéines et des acides nucléiques chez Bacillus cereus

In the presence of 8-azaguanine, protein synthesis is strongly inhibited and the formation of constitutive penicillinase is completely abolished. Low concentrations of azaguanine which are sufficient to block penicillinase formation do not change the rate of DNA synthesis. Higher concentrations cause an initial slight inhibition which becomes much stronger after about one doubling of the DNA. Azaguanine increases the rate of RNA formation while being incorporated into RNA. At low concentration the analogue is incorporated for a limited time only; later a large part of it is rejected from the RNA. The RNA of bacteria which have incorporated azaguanine is more labile in acid medium than normal RNA. The effects of azaguanine on B. cereus resemble those of chloramphenicol on E. coli and S. aureus.

Metabolic changes in nucleic acids during the induction of enzymes by oxygen in resting yeast

Abstract The formation of catalase and of several other enzymes which are induced by oxygen in a respiratory-deficient mutant of yeast is accompanied by an increased incorporation of labeled adenine and uracil into RNA and DNA. Different RNA fractions isolated by ammonium sulfate fractionation do not show the same relative increase of adenine incorporation during adaptive enzyme formation. The results reported indicate that in resting yeast enzymatic adaptation is associated with changes in some RNA fractions and possibly in DNA.

Effect of proflavine on the binding of isoleucine to transfer RNA

Abstract Proflavine (3,6-diaminoacridine) reduces the amount of isoleucine which can be enzymically bound to tRNA in vitro . The activation of isoleucine by isoleucyl-sRNA synthetase ( l -isoleucine: sRNA ligase (AMP), EC 6.1.1.5), as measured by ATP-pyrophosphate exchange in the absence of sRNA, is not affected by the compound. Evidence for the strong binding of proflavine to sRNA is presented, and some characteristics of the RNA-proflavine complex are described. The binding of proflavine to tRNA may explain the reduction of isoleucyl-sRNA formation.

Restauration de la synthèse d'enzymes après inhibition par l'azaguanine

Abstract Azguanine inhibits protein synthesis almost completely. The inhibition can be released by guanosine. The later guanosine is added, the more imperfect becomes the restoration of protein synthesis. The damages caused by azaguanine affect more severely the formation of certain enzymes than that of others. Is is conculded that azaguanine interferes with the activity of substances which are involved in the control of specificity in protein synthesis.

Formation induite de catalase et métabolisme des acides nucléiques chez la levure effet des rayons X

Abstract Effect of X-rays on the induced catalase formation and the metabolism of nucleic acids in yeast X-rays (105 R) do not prevent the synthesis of catalase in resting yeast; they can induce or stimulate the formation of the enzyme. Doses of X-rays which damage DNA in vivo to the point that it is easily split into small molecules do not inhibit the synthesis of catalase. The effects of X-rays on the synthesis of RNA and DNA which accompanies the induced formation of enzymes are discussed.

Biochemical features of bovine leukemia virus

Abstract Short term cultures of bovine leukemic lymphocytes (enzootic form of the disease) release virus particles with biochemical properties of RNA oncogenic viruses. These particles, called Bovine Leukemia Virus (BLV), are never produced by normal lymphocytes. They have a high molecular weight-reverse transcriptase complex and a density averaging 1.155 g/ml in sucrose solutions. Molecular hybridizations between BLV 3HcDNA and several viral RNAs show that BLV is not related to Mason-Pfizer Monkey Virus (MPMV), Simian Sarcoma Associated Virus (SSV-1), Feline Leukemia Virus (FeLV) or Avian Myeloblastosis Virus (AMV). Rauscher Leukemia Virus (RLV) exhibits a slight but reproducible relatedness to BLV. These first results were confirmed by hybridizing 3H cDNA made in the above viruses to BLV 7OS RNA. In tumor DNA (enzootic form of the disease) 125I RNA hybridization experiments, Fe LV and AMV 7OS RNA did not compete with BLV RNA. DNA-DNA annealing reactions using BLV 3H cDNA as a probe strongly suggest that the DNA of bovine leukemic cells (enzootic leukemia) contains viral sequences that cannot be detected in normal bovine DNA. We are thus lead to consider BLV as an exogenous (class II) tumor virus.