Fluorescent study of tobacco mosaic virus protein

The synthesis of two new variants of gramicidin is described. It is shown that the changes in the aromatic side groups do not influence the single channel conductivity. Experiments in which solutions having different molarities on the two sides of the bilayer lipid membrane are described and their results compared with a rate theory analysis. It is concluded that the gramicidin pore contains approximately 10 equal potential maxima.     

Suitable conditions for characterization,identification, and isolation of the mRNA of the small subunit of ribulose-1,5-bisphosphate carboxylase from Nicotiana sylvestris

The products synthesized in vitro by messenger RNA (mRNA) extracted from Nicotiana sylvestris were analyzed by electrophoresis on polyacrylamide slab gels. Only three of the major polypeptides synthesized are considered here: P₅₅, P₃₂, and P₂₀. P₅₅ and P₃₂ were translated from chloroplast mRNA. P₅₅ corresponds to the large subunit of ribulose-1,5-bisphosphate (RuP₂) carboxylase; P₃₂ is probably a chloroplast membrane protein. P₂₀, the polypeptide synthesized from cytoplasmic poly(A)⁺ RNA, is the precursor of the small subunit of RuP₂ carboxylase. The balance between P₂₀ and P₃₂, in which their relative proportions varied inversely, was regulated by the age of the leaves and the time of illumination; we took advantage of this phenomenon to isolate the mRNA from the small subunit in relatively large amounts. This mRNA has a molecular weight of 350,000.Abbreviations RuP₂ ribulose-1,5-bisphosphate - mRNA messenger RNA - SDS sodium dodecyl sulfate     

Monocistronic translation of alfalfa mosaic virus RNAs.

The four alfalfa mosaic virus RNAs (respectively 24 S, 20 S, 17 S and 12 S) have been used separately as messengers in two in vitro protein synthesizing systems: wheat germ and rabbit reticulocyte lysate. In both systems a polypeptide corresponding to the translation of the entire length of the RNA can be found for RNAs 24 S, 20 S and 12 S, but not for 17 S RNA, the translation product of which is only 35,000 daltons. The number of initiation sites has been determined for each RNA by analyzing the initiation peptides synthesized in the presence of spasomycin and show that there is only one initiation or binding site perRNA. We thus conclude that each AMV RNA behaves as a monocistronic messenger in in vitro translating systems.     

Identification of a novel amino acid alpha-tyrosine 93 within the cholinergic ligands-binding sites of the acetylcholine receptor by photoaffinity labeling. Additional evidence for a three-loop model of the cholinergic ligands-binding sites

The native, membrane-bound, acetylcholine receptor from Torpedo marmorata was photolabeled by the competitive antagonist p-[3H]dimethylaminobenzene-diazonium fluoroborate (DDF) in the presence of the noncompetitive blocker phencyclidine and under energy transfer conditions. The isolated alpha-subunits were treated with cyanogen bromide and fractionation of the resulting fragments yielded three radiolabeled peptides, at the level of which, incorporation of [3H]DDF (i) was equally inhibited by the agonist carbamoylcholine and the competitive antagonist alpha-bungarotoxin and (ii) was insensitive to "scavenging" reagents. Subfragmentation of cyanogen bromide peptide III with omicron-iodosobenzoic acid or trypsin and sequence analysis of the fragments led to the identification of a novel amino acid alpha-Tyr-93 (and possibly Trp-86) as labeled by [3H]DDF in a carbamoylcholine-sensitive manner. alpha-Tyr-93 is conserved in the muscle and neuronal alpha-subunits but not in the other subunits of muscle receptor. This result provides evidence for a site involving at least a third loop of the alpha-subunit amino-terminal hydrophilic domain, in addition to the ones previously identified (Dennis, M., Giraudat, J., Kotzyba-Hibert, F., Goeldner, M., Hirth, C., Chang, J. Y., Lazure, C., Chretien, M., and Changeux, J. P. (1988) Biochemistry 27, 2346-2357). Possible contribution of tyrosine side-chains to the complexation of the quaternary ammonium group of cholinergic ligands is discussed.     

Thin-layer separation and quantification of bile acids

A method has been developed for quantification of total free and conjugated bile acids separated on silica gel HR coated thin-layer chromatography plates. Aliquots of bile acid solutions are applied to channeled plates which are developed with either ethyl acetate: isooctane: glacial acetic acid 10:10:2 v/v for free bile acid separation, or chloroform:methanol:glacial acetic acid:water 130:50:4:8 v/v for conjugated bile acid separation. Bile acids are determined directly in serial areas of silica gel by treating gel areas suspended in tris buffer with resazurin reagent. The method is quantitative and as little as 0.1 μg of bile acid is readily determined. Application of the method to determinations of bile acids in crude fecal extracts is described.     

DNA sequences at the sites of three insertions of the transposable element Tn5 in the histidine operon of Salmonella

Summary The DNA sequence has been determined at the sites of three independent occurrences of the transposable element Tn5 in the hisG gene of the histidine operon. All three insertion sites are adjacent to G+C-rich stretches of DNA. In all three cases sequences at the sites of insertion show homology with a region near the ends of Tn5.     

Teratologic evaluation of synthetic delta 9-tetrahydrocannabinol in rabbits.

Synthetic delta 9-tetrahydrocannabinol (THC) was dissolved in undiluted propylene glycol and administered in daily subcutaneous doses of 15.0, 30.0 or 60.0 mg/kg to pregnant New Zealand white rabbits on days 7--19 of gestation. Maternal food consumption and weight gain were markedly reduced at all dose levels. Embryotoxicity and embryocidal effects were observed in the form of reduced litter weight and number of viable fetuses, respectively, in offspring from pregnant mothers treated with THC. However, on the basis of extensive external, visceral and skeletal examination of all fetuses it may be concluded that THC is not teratogenic in the New Zealand white strain rabbit following subcutaneous administration of doses as high as 60.0 mg/kg/day during the critical period of organogenesis (days 7--19 of gestation). On the other hand, an oral dose of thalidomide (200.0 mg/kg/day), the positive control used in this study, was both embryocidal and teratogenic.     

Nature and specificity of the RNA-protein interaction in the case of the tymoviruses.

Dissociation-reassociation experiments performed with turnip yellow mosaic virus in the presence of various RNAs and polynucleotides were used to investigate the degree of specificity and the contribution of the associated RNA moiety to the stability of TYMV. The results emphasize the importance of strategic cytosine residues spread along the RNA chain. Some insight into the contribution of the protein could be gained from comparison of TYMV and eggplant mosaic virus (EMV), a virus similar to TYMV although its top component contains low molecular mass RNA's able to bind various amino acids. Hydrophobic interactions between protein subunits are less important in EMV than in TYMV, and artificial capsids could be obtained from dissociated EMV coat protein. Whether the capsid is or is not the precursor of the virion in tymovirus morphogenesis is discussed.     

Some properties of tobacco protoplast chromatin.

Chromatin was prepared from tobacco-leaf protoplasts. Its solubility in increasing molarities of NaCl was studied and the structure of the soluble fraction observed by electron microscopy. We demonstrate that in plants, the DNA and histones are associated in beaded structures similar to those called omicron-bodies or nucleosomes in animal chromatin. The nucleosomes were associated with DNA in either compact or extended forms. The compact arrangement was predominant in the fraction solubilized between 0.1 and 0.4 M NaCl. The extended form, present at 0.5 and 0.6 M NaCl. showed DNA filaments of various lengths interspacing the nucleosomes. At these ionic strengths ring structures were present, associated with the DNA. At 0.7 M NaCl and above, only DNA filaments were present, occasionally associated with big rings, and nucleosomes were compoetely dissociated. Free DNA molecules were present at all ionic strengths used. The possible origin and significance of the rings are discussed.