Biochimica et Biophysica Acta (BBA) - Specialized Section on Nucleic Acids and Related Subjects最新文献

Lysyl-s-RNA synthetase (l-lysine: s-RNA ligase (AMP), EC 6.1.1.6) and methionyl-s-RNA-synthetase (l-methionine: s-RNA ligase (AMP), EC 6.1.1.10) were purified from wheat germ about 600-fold and 160-fold, respectively. The two enzymes catalyse amino acid-dependent ATP-pyrophosphate exchange as well as the incorporation of amino acids into s-RNA. The pH optimum of lysine enzyme is in the range 6.8–7.1 and that of methionine enzyme in the range 8.1–8.5. Both enzymes are strongly inhibited by p-hydroxymercuribenzoate and the activity is restored by sulfhydryl group-containing compounds.

The study of the sequence of purine nucleotides in deoxyribonucleic acids requires the availability of suitable preparations of apyrimidinic acid, i.e., of polydeoxyribophosphate chains retaining the purines of the parent DNA in unchanged sequence and proportions. For an understanding of the degradation of DNA a detailed knowledge and a complete balance of the hydrazinolysis of pyrimidine deoxyribomononucleotides were required.

The course and the rate of the degradation by hydrazine of thymidylic and deoxycytidylic acids were studied under a variety of conditions. These nucleotides, when treated with anhydrous hydrazine, give rise to stoichiometric amounts of (a) urea; (b) the hydrazone of 2-deoxyribophosphate; and (c) a heterocyclic compound. The latter is 4-methyl-5-pyrazolone in the case of thymidylic acid, and 3(5)-amino-pyrazole in the case of deoxycytidylic acid. The hydrazone of the sugar phosphate is cleaved quantitatively with benzaldehyde to yield 2-deoxyribophosphate and benzalazine. The various breakdown products were identified by comparison with authentic preparations.

The first-order reaction of the degradation with anhydrous hydrazine is completed at 60° within 1.5 h for deoxycytidylic acid and within 4 h for thymidylic acid. At 40°, the time required for completion is about 4 times as long.

Other orienting experiments demonstrated that purine deoxyribonucleotides withstand the treatment with hydrazine; that aqueous hydrazine solutions at various pH values effect a slower and irregular degradation; and that the replacement of hydrazine by monosubstituted hydrazine derivatives, such as phenyl- or methylhydrazine does not yield the desired results.

• 1.

  1. DNA preparations from Euglena chloroplasts were found to contain a major component with a density of 1.684 in addition to the nuclear component with a density of 1.708. Partial lysis of the chloroplasts resulted in the removal of the nuclear component. A component with a density of approx. 1.692 was also associated with the chloroplast preparations.

• 2.

  2. The temperature of melting of the chloroplast DNA was 78–80°, as compared to 89–91° for the nuclear DNA.

• 3.

  3. Nucleotide analysis of the chloroplast DNA indicates an overall G+C content of about 25 mole%. The 5-methylcytosine of Euglena DNA is concentrated in the nuclear material.

• 4.

  4. The amount of DNA per chloroplast appears to be about the same as that in an Escherichia coli cell.

• 5.

  5. The nucleotide composition of the ribosomal RNA of Euglena chloroplasts seems to be influenced by that of the chloroplast DNA.

The rate of synthesis of hemoglobin in a cell-free system of rabbit reticulocytes is increased by the addition of RNA from rabbit liver, kidney and intestine. This stimulation involves neither transfer RNA, nor a release factor, nor a protection of endogenous RNA. The evidence that this stimulation really involves hemoglobin synthesis is based on the isolation of purified hemoglobin, on the comparison of the rate of incorporation of several labelled amino acids and also on the comparison of the effect of addition of RNA on the incorporation of these amino acids. The RNA's from guinea-pig, rat, mouse and chicken livers, are either inactive or inhibitory on the cell-free synthesis of rabbit hemoglobin. The problem of the existence of hemoglobin-specific messenger RNA in non-hematopoietic cells is discussed in relation with the cell differentiation of protein biosynthesis.

The RNA-like product obtained by incubation of ATP, GTP, UTP, and CTP with a cell-free extract from tobacco leaves has been purified by phenol extraction and passage through Sephadex. When either [α-³²P]ATP or [α-³²P]GTP was included in the reaction mixture the major portion of the incorporated counts remained associated with the RNA during purification. In both cases, alkaline hydrolysis of this purified, labeled product yielded AMP, GMP, CMP, and UMP all of which were ³²P-labeled. This provides direct evidence that the labeled product of the reaction contains all four nucleotides linked by interribonucleotide bonds to form a true RNA.

The sensitive RNA bacteriophages MS2 and f2 and, to a lesser extent, the single-stranded DNA phage S13 are inactivated when their dilute suspensions come into contact with an aluminum alloy surface or when diluted with fluids which have been in contact with aluminum, zinc, or magnesium. The inactivation is believed to result from the simultaneous action of traces of Cu²⁺ and electrolytically formed H₂O₂ and may be stimulated by addition of both of these agents, although neither alone is fully active when present in trace amounts. The phages are protected by adding either catalase or EDTA, which is further support for the suggested mechanism.

The damages produced by X-rays do not prevent either denaturation or renaturation of DNA. It was possible to prepare hybrid molecules with an X-ray-inactivated DNA carrying a genetic marker (streptomycin resistance) and a wild-type DNA. We have examined the rescue of transforming activity of the irradiated DNA, in respect of the irradiation dose and of the concentration ratio of the two DNA's at the time of the renaturation.