Homogeneity among Senegalese strains of yellow fever virus.

A series of 16 yellow fever (YF) viruses isolated from mosquitoes, monkeys and humans in different epidemiological contexts in Senegal and The Gambia between 1976 and 1983, was analyzed by T1 RNase oligonucleotide fingerprints of the genomic 32P-labeled RNA, by SDS-polyacrylamide gel electrophoresis of the intracellular virus-specified polypeptides, by peptide mapping of the envelope E glycoprotein and by immunological reactivities with monoclonal antibody fluids (MAF's) against the E glycoprotein. These strains had not been passed in suckling mice and were isolated in Aedes pseudoscutellaris Mos 61 cultured cells. These strains showed no virulence in three-week-old Swiss mice when injected intraperitoneally. Direct comparison of the large T1 RNase-resistant oligonucleotide maps indicated a relative genetic stability (92%-100%). A greater change was observed when these strains were compared with an epidemic YF strain isolated in 1965 with an oligonucleotide fingerprint map sharing 82%-88% similarity. The YF-specified proteins were identical in their molecular weight, and the fragments obtained after limited proteolysis of the envelope protein using protease V8 or alphachymotrypsine indicated that the strains were chemically similar. Only a few differences were observed when the strains were seroneutralized with MAF's, but no relation could be made with genetic or biological data. This suggested that the YF virus strains isolated from the same geographic area and during a short period of time had evolved slowly. Moreover, all the viruses were closely related and no correlation could be established with the apparent variations in virulence in nature.

Association of an aminoacyl-tRNA synthetase complex and of phenylalanyl-tRNA synthetase with the cytoskeletal framework fraction from mammalian cells.

The intracellular distribution of several mammalian aminoacyl-tRNA synthetases was investigated by biochemical and immunocytological approaches. The fraction of amino-acyl-tRNA synthetases bound to the detergent-insoluble cytoskeletal framework obtained after extraction of NRK cells by 0.1% Triton X-100 was estimated, by activity measurements, to about 80% for phenylalanyl-tRNA synthetase and 40% for the high-molecular-weight (HMW) complex containing the seven aminoacyl-tRNA synthetases specific for glutamic acid, isoleucine, leucine, methionine, glutamine, lysine, and arginine. This association was shown to be salt-dependent. The subcellular localization of these enzymes was examined using an immunocytological approach. When cultured cells were fixed with paraformaldehyde and then permeabilized with Triton X-100, a fairly uniform cytoplasmic labelling was observed with antibodies directed to the aminoacyl-tRNA synthetase complex or to phenylalanyl-tRNA synthetase. By contrast, when cells were extracted with 0.1% Triton X-100 prior to fixation with paraformaldehyde, the staining patterns obtained with antibodies to aminoacyl-tRNA synthetases were very similar to that obtained with antibodies to rough endoplasmic reticulum, as assessed by single or double indirect immunofluorescence microscopy. These results suggest that free and bound forms of these aminoacyl-tRNA synthetases may coexist within the cell. In addition to cytoplasmic labelling, antibodies directed to phenylalanyl-tRNA synthetase stained the nucleus of rapidly growing cells. The possible significance of this finding is discussed.

Phenylalanyl-tRNA synthetases from sheep liver and yeast. Correlation between net charge and binding to ribosomes.

Unlike phenylalanyl-tRNA synthetase from lower eukaryotes, the corresponding enzyme from higher eukaryotes displays a pronounced tendency to associate with ribosomes in vitro. To attempt to uncover the structural features responsible for this difference in behavior, a comparative study of the enzymes purified to homogeneity from sheep liver and yeast was undertaken. The two alpha 2 beta 2-type enzymes displayed remarkably similar subunit molecular masses (71 and 63 kDa for sheep, 74 and 63 kDa for yeast), yet differed markedly in their isoelectric points (8.0 and 5.6 pH units, respectively). Mild tryptic digestion of the enzyme from sheep led to preferential degradation of the 63-kDa beta subunit into two major fragments of 35 and 25 kDa, respectively, with concomitant loss of activity. The isoelectric points of the denatured fragments were found to be distinctly lower than that of the denatured beta subunit, implying that the residues responsible for the basic net charge of the original beta subunit are mainly clustered in a small portion of the polypeptide chain which was excised during proteolysis. Despite their different isoelectric points, the enzymes from yeast and sheep displayed identical requirements for aminoacylation of tRNA at optimal rates. Moreover, the incidence of variations in pH and ionic strength on the kinetic parameters of the two enzymes was indistinguishable. Interpreted in terms of the polyelectrolyte theory, these results support the view that the residues responsible for the basic net charge of the mammalian enzyme are located in a region distal from the active site. It is suggested that the cationic charge of the enzyme allows anchorage to a cellular component carrying negative charges, possibly the ribosome.

Sedimentation behaviour of aminoacyl-tRNA synthetases from mixed lysates of yeast and rabbit liver.

The subcellular distribution of five aminoacyl-tRNA synthetases from yeast, including lysyl-, arginyl- and methionyl-tRNA synthetases known to exist as high-molecular-weight complexes in lysates from higher eukaryotes, was investigated. To minimize the risks of proteolysis, spheroplasts prepared from exponentially grown yeast cells were lysed in the presence of several proteinase inhibitors, under conditions which preserved the integrity of the proteinase-rich vacuoles. The vacuole-free supernatant was subjected to sucrose density gradient centrifugation. No evidence for multimolecular associations of these enzymes was found. In particular, phenylalanyl-tRNA synthetase activity was not associated with the ribosomes, whereas purified phenylalanyl-tRNA synthetase from sheep liver, added to the yeast lysate prior to centrifugation, was entirely recovered in the ribosomal fraction. A mixture of lysates from yeast and rabbit liver was also subjected to sucrose gradient centrifugation and assayed for methionyl- and arginyl-tRNA synthetase activities, under conditions which allowed discrimination between the enzymes originating from yeast and rabbit. The two enzymes from rabbit liver were found to sediment exclusively as high-molecular-weight complexes, in contrast to the corresponding enzymes from yeast, which displayed sedimentation properties characteristic of free enzymes. The preservation of the complexed forms of mammalian aminoacyl-tRNA synthetases upon mixing of yeast and rabbit liver extracts argues against the possibility that failure to observe complexed forms of these enzymes in yeast was due to uncontrolled proteolysis. Furthermore, this result denies the presence, in the crude extract from liver, of components capable of inducing artefactual aggregation of the yeast aminoacyl-tRNA synthetases, and thus indirectly argues against an artefactual origin of the multienzyme complexes encountered in lysates from mammalian cells.

Zinc-dependent synthesis of 5',5'-diadenosine tetraphosphate by sheep liver lysyl- and phenylalanyl-tRNA synthetases.

Zinc greatly stimulates the initial rate of in vitro synthesis of 5',5'-diadenosine tetraphosphate by sheep liver lysyl- and phenylalanyl-tRNMA synthetases. In the case of each enzyme, maximum stimulation of 50- to 100-fold is reached upon addition of 50 microM ZnC2 to a reaction mixture (37 degrees c, pH 7.8) containing 50 microM dithioerythritol, 150 mM KCl, 5 mM ATP, 10 mM MgCl2, 0.1 mM cognate L-aminoacid, catalytic amounts of the aminoacyl-tRNA synthetase, and unlimiting pyrophosphatase activity. This observation made with aminoacyl-tRNA synthetases of mammalian origin supports the proposal that changes in cellular free zinc ion concentration could contribute to 5',5'-diadenosine tetraphosphate variations in animal cells as a function of growth activity.

Macromolecular complexes from sheep and rabbit containing seven aminoacyl-tRNA synthetases. I. Species specificity of the polypeptide composition.

Using a three-step procedure designed to minimize the risks of proteolysis, high molecular weight complexes containing the same seven aminoacyl-tRNA synthetases specific for isoleucine, leucine, methionine, lysine, arginine, glutamic acid, and glutamine were purified from sheep liver and spleen, as well as from rabbit reticulocytes and liver. The polypeptide composition of these complexes, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is characteristic of the animal species from which they are derived. The complexes from sheep liver and spleen display indistinguishable polypeptide patterns composed of 11 major components. Of the 10 common components which characterize the complexes of rabbit reticulocytes and liver, 4 are also shared by the complexes from sheep, while 6 have distinctly different electrophoretic mobilities. Furthermore, in the case of the complex from rabbit reticulocytes, it is shown that the enzyme and polypeptide composition of the complex is independent of the purification method employed. The isolation of high molecular weight complexes of identical aminoacyl-tRNA synthetase and polypeptide compositions from two cell types as radically different as rabbit reticulocytes and hepatocytes suggests that these multienzyme complexes do not arise as artifacts of preparation and supports the view that they reflect a structural organization existing within the cell.