Effect of chemical carcinogens and partial hepatectomy on "in vivo" (35S)methionine interaction with rat liver tRNA

The effect of carcinogens given by a single or multiple injections on the extent of (35S)methionine interaction with hepatic tRNA was studied in normal and partially hepatectomized rats. Either partial hepatectomy or administration of ethionine (100 or 330 mg/kg body weight) and dimethylnitrosamine (120 mg/kg body weight) by multiple i.p. injections inhibited the (35S)methionine-tRNA interaction, while administration of hepatocarcinogenic chemicals plus PH resulted rather in a stimulation. Methylnitrosourea enhanced the extent of interaction when administered in a single dose (100 mg per kg body weight) 18 h after partial hepatectomy.     

"In vivo" (35S)-methionine interaction with rat-liver DNA and the effect of chemical carcinogens

1. After intraperitoneal administration of (35S)methionine (25 mg, 1.6 mCi/kg), detectable amount of radioactivity resulted associated to rat-liver DNA: the interaction reached the maximum value (about 18 pmol/mumol DNA P) by 2 h after administration of radioactive aminoacid. 2. The (35S)-binding was inhibited by the hepatocarcinogenic ethionine and dimethylnitrosamine, and was stimulated by the non-hepatocarcinogenic methylnitrosourea. 3. Hplc analysis of (35S)DNA enzymic digest evidenced two radioactive compounds, the UV behaviour of which is reported.     

Non-acylated tRNA binding on rat liver 60S subunits

The ability of rat liver ribosomes and subunits to form complexes with non-acylated tRNAs in the absence of mRNA has been studied using nitrocellulose membrane filtration technique. Binding to 60S subunits required the integrity of the pCpCpA end of the tRNA molecule and was not decreased when unpaired guanine had been modified using kethoxal. Scatchard plot analysis suggests that large subunits have two binding sites, whose affinity constant values, relatively high, vary according to the ionic composition of the medium. Thus, the affinity constant of the stronger site (about 3. 10⁹ M^?1) is from 7 to 21 times higher than that of the weaker. High Mg²⁺ and low K⁺ concentrations stabilized binding to both sites. tRNA is at least partly retained on the subunits by heat-labile bonds.     

Labelling of proteins with [35S]methionine in monolayer cultures of rat hepatocytes

Optimal conditions for the labelling of proteins with [35S]methionine in monolayers of rat hepatocytes have been established. The ability to incorporate the radioactive amino acid was constant for at least 26 h and independent of whether the medium was buffered with CO2/HCO3 or with 4-(2-hydroxyethyl)-1-piper-azineethanesulphonic acid (Hepes). Preincubation in methionine-free medium for up to 30 min yielded increasing, and from 60 to 180 min decreasing, rates of incorporation. An apparent Km value of 0.06 mM was obtained for the incorporation reaction in cells preincubated for 40 min.     

tRNA binding stabilizes rat liver 60 S ribosomal subunits during treatment with LiCl

We have shown recently that, in the absence of mRNA, 1 molecule of nonacylated tRNA binds to the large ribosomal subunit of rat liver with a high affinity constant (Buisson, M., Reboud, A.M., Dubost, S., and Reboud, J. P. (1979) Biochem. Biophys. Res. Commun. 90,634-640). In this paper, free and tRNA-bound 60 S subunits were treated with increasing concentrations of LiCl to obtain information on tRNA binding site. The rationale for using deacylated tRNA was that it is assumed to bind to the peptidyl donor site. We observed that tRNA has a strong protective effect on subunit modifications produced by LiCl: tRNA prevents subunit inactivation as measured by puromycin reaction and polyphenylalanine synthesis and it shifts the Li+/Mg2+ ratio value needed to reach 50% inactivation, from 60 to 250; it also prevents ribosomal protein and 5 S RNA release and large sedimentation changes of subunits, induced by LiCl. To explain the mechanism of 60 S subunit stabilization by tRNA, two hypotheses are considered: stabilization can be consequent on direct interaction of tRNA with specific proteins, or on maintenance on subunits of essential cations which are otherwise displaced by Li+, or both.     

Conversion of a methionine initiator tRNA into a tryptophan-inserting elongator tRNA in vivo.

The role of the anticodon and discriminator base in aminoacylation of tRNAs with tryptophan has been explored using a recently developed in vivo assay based on initiation of protein synthesis by mischarged mutants of the Escherichia coli initiator tRNA. Substitution of the methionine anticodon CAU with the tryptophan anticodon CCA caused tRNA(fMet) to be aminoacylated with both methionine and tryptophan in vivo, as determined by analysis of the amino acids inserted by the mutant tRNA at the translational start site of a reporter protein containing a tryptophan initiation codon. Conversion of the discriminator base of tRNA(CCA)fMet from A73 to G73, the base present in tRNA(Trp), eliminated the in vivo methionine acceptor activity of the tRNA and resulted in complete charging with tryptophan. Single base changes in the anticodon of tRNA(CCA)fMet containing G73 from CCA to UCA, GCA, CAA, and CCG (changes underlined) essentially abolished tryptophan insertion, showing that all three anticodon bases specify the tryptophan identity of the tRNA. The important role of G73 in tryptophan identity was confirmed using mutants of an opal suppressor derivative of tRNA(Trp). Substitution of G73 with A73, C73, or U73 resulted in a large loss of the ability of the tRNA to suppress an opal stop codon in a reporter protein. Base pair substitutions at the first three positions of the acceptor stem of the suppressor tRNA caused 2-12-fold reductions in the efficiency of suppression without loss of specificity for aminoacylation of the tRNA with tryptophan.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo interaction between mouse liver lysosomes and chloroquine

Particles sedimenting at 27,000 g X 10 min (MLCQ) were separated from liver homogenates of mice injected with chloroquine (CQ). The MLCQ contained most of the drug recovered in the organ as well as 50% of the liver aryl sulphatase activity. The release of CQ from MLCQ was studied in some physicochemical conditions, and in the presence of various agents known to modify membrane composition and stability. At pH 7.4, the equilibrium between free and bound CQ depended on the dilution of the MLCQ, and the time to reach equilibrium was strongly influenced by the temperature of incubation. Several agents causing membrane disruption and lysosomal enzyme leakage, such as osmotic shock, sonication and digitonin, had little effect on the CQ release. Acid and alkaline buffers, 0.55 M KCl and 0.1% Triton X-100 caused, instead, the immediate release of most of the bound CQ. Concentrations of digitonin causing the release of aryl sulphatase activity had little effect on bound CQ, suggesting that the drug is retained in lysosomes by forces and/or structures different in nature from those retaining most of the lysosomal enzyme activity. We think that the CQ trapped in lysosomes is bound to high affinity sites in membranous structures which are particularly altered by agents known to extract peripheral proteins from biological membranes or to change the conformation of molecular structures.     

In vivo misreading by tRNA overdose

Rpb5-H147R is an AT-GC transition replacing CAC(His) by CGC(Arg) at a conserved and critical position of ABC27 (Rpb5p), one of the five common and essential subunits shared by all three eukaryotic RNA polymerases. This mutation is viable at 25 degrees C, but has a lethal phenotype at 34 degrees C. A search for dosage-dependent suppressors identified five distinct clones that all bear a copy of the tRNA(His)GUG gene. Suppression was also observed with a small genomic insert bearing this tRNA gene and no other coding sequences, under conditions where there is a sevenfold increase in the cellular concentration of tRNA(His)GUG. Overexpressing tRNA(Arg)ICG, which normally decodes the suppressed CGC codon, counteracted suppression. Suppression is codon specific because it was abolished when replacing CGC by its synonymous codons CGA, CGU, or AGA, but was not detectably affected by several nucleotide substitutions modifying the surrounding sequence and is thus largely insensitive to the nucleotide context. It is proposed that overexpressing tRNA(His)GUG extends its decoding properties from CAC(His) to the noncognate CGC(Arg) codon through an illegitimate U x G pairing at the middle base of the anticodon. Accordingly, tRNA(His)GUG would compete with tRNA(Arg)ICG for chain elongation and generate a significant level of misreading errors under normal growth conditions.     

Preparative labeling of proteins with [35S]methionine.

The most common technique for preparative labeling of proteins with radioisotopes for experimental purposes utilizes 125I. This isotope has certain limitations, including the emission of gamma- and X-irradiation, the release of gaseous 125I2 from solutions of Na 125I, and the potential for concentration of 125I in thyroid glands. We have discovered a means for labeling proteins rapidly and simply with [35S]methionine. The technique is applicable to a wide variety of proteins. Antibodies labeled by our technique remain functional.     

In vitro conversion of a methionine to a glutamine-acceptor tRNA

A derivative of Escherichia coli tRNAfMet containing an altered anticodon sequence, CUA, has been enzymatically synthesized in vitro. The variant tRNA was prepared by excision of the normal anticodon, CAU, in a limited digestion of intact tRNAfMet with RNase A, followed by insertion of the CUA sequence into the anticodon loop with T4 RNA ligase and polynucleotide kinase. The altered methionine tRNA showed a large enhancement in the rate of aminoacylation by glutaminyl-tRNA synthetase and a large decrease in the rate of aminoacylation by methionyl-tRNA synthetase. Measurement of kinetic parameters for the charging reaction by the cognate and noncognate enzymes revealed that the modified tRNA is a better acceptor for glutamine than for methionine. The rate of mischarging is similar to that previously reported for a tryptophan amber suppressor tRNA containing the anticodon CUA, su+7 tRNATrp, which is aminoacylated with glutamine both in vivo and in vitro [Yaniv, M., Folk, W. R., Berg, P., & Soll, L. (1974) J. Mol. Biol. 86, 245-260; Yarus, M., Knowlton, R. E., & Soll, L. (1977) in Nucleic Acid-Protein Recognition (Vogel, H., Ed.) pp 391-408, Academic Press, New York]. The present results provide additional evidence that the specificity of aminoacylation by glutaminyl-tRNA synthetase is sensitive to small changes in the nucleotide sequence of noncognate tRNAs and that uridine in the middle position of the anticodon is involved in the recognition of tRNA substrates by this enzyme.     

The uptake of labelled sulphur by the proteins of human liver slices incubated with L-(35S)-methionine or (35S)-sulphate

Transfer of labelled sulphur from methionine into protein-bound cysteine was observed in human foetal liver $\text{in}$$\text{vitro}$. Inorganic sulphate could not be utilized as sulphur donor for cystein synthesis in human foetal liver.     

In vivo and in vitro investigation of bacterial type B RNase P interaction with tRNA 3'-CCA

For catalysis by bacterial type B RNase P, the importance of a specific interaction with p(recursor)tRNA 3'-CCA termini is yet unclear. We show that mutation of one of the two G residues assumed to interact with 3'-CCA in type B RNase P RNAs inhibits cell growth, but cell viability is at least partially restored at increased RNase P levels due to RNase P protein overexpression. The in vivo defects of the mutant enzymes correlated with an enzyme defect at low Mg(2+) in vitro. For Bacillus subtilis RNase P, an isosteric C259-G(74) bp fully and a C258-G(75) bp slightly rescued catalytic proficiency, demonstrating Watson-Crick base pairing to tRNA 3'-CCA but also emphasizing the importance of the base identity of the 5'-proximal G residue (G258). We infer the defect of the mutant enzymes to primarily lie in the recruitment of catalytically relevant Mg(2+), with a possible contribution from altered RNA folding. Although with reduced efficiency, B. subtilis RNase P is able to cleave CCA-less ptRNAs in vitro and in vivo. We conclude that the observed in vivo defects upon disruption of the CCA interaction are either due to a global deceleration in ptRNA maturation or severe inhibition of 5'-maturation for a ptRNA subset.     

In vitro interaction of 63-nickel(II) with chromatin and DNA from rat kidney and liver nuclei

The interaction of nickel(II) with chromatin was studied in vitro and in isolated nuclei from rat liver and kidney. Nickel(II) bound to chromatin, polynucleosomes (DNA + histone octamer protein complex), and to deproteinized DNA both in intact nuclei and in vitro. The amount of nickel(II) bound depended on the concentration of nickel(II), the presence of chromosomal proteins and the binding sites on DNA which provide a stable coordination environment for nickel(II). The binding of nickel(II) to chromatin and to DNA in whole nuclei was much slower than in vitro indicating that assessibility of the DNA binding sites was influenced by the presence of the nuclear membrane, nuclear matrix and nuclear proteins and/or by the condensed nuclear structure of chromatin. Since DNA containing bound nickel(II) was isolated from chromatin, nickel(II) directly interacted with stable binding sites on the DNA molecule in chromatin. Nickel(II) was associated with the histone and non-histone nuclear proteins as well as the DNA in rat liver and kidney chromatin. Nickel(II) was found to bind to calf thymus histones in vitro. Nickel(II)-nuclear protein and -DNA interactions were investigated by gel electrophoretic analysis of in vitro incubation products. Although nickel-histone and nickel-non-histone protein interactions were completely disrupted by the electrophoretic conditions, fluorography revealed the presence of inert nickel(II)-DNA and/or nickel(II)-DNA-protein complexes.