Auto-radiographic determination of S35 in tissues after injection of methionine-S35 and sodium sulfate-S35

The loss of "bound" S(35) that occurs during various mounting procedures used in autoradiography was studied in healing surface wounds of rats treated with either methionine-S(35) or Na(2)S(35)O(4). Valid autoradiography of bound S(35) in this tissue is not possible until 48 hours after radiosulfate and 24 hours after radiomethionine injection, when the S(35) is almost entirely bound in large protein and polysaccharide molecules. Autoradiograms of S(35) given in both the organic and inorganic form reveal substantial over-all loss of the bound isotope from sections subjected to contact with solvents prior to autoradiography. A comparison of autoradiograms prepared by dry-mounting sections of frozen-dried tissue with autoradiograms of wet-mounted sections of the same tissue suggest that the loss is proportional to the extent of the contact with solvents. Evidence suggests that loss of the isotope occurs during contact of the ribbon or section itself with solutions after fixation and cutting and prior to radiation exposure. No appreciable loss of the bound isotope seems to occur during contact of the intact tissue specimen with a variety of fluid fixatives except for a marginal zone at the excision edges of the tissue. The potential hazard of displacement of the isotope during fixation, however, remains. Technics which prevent loss of the isotope and fogging of the nuclear emulsion permit the use of thinner sections and emulsion films and the fine resolution of image rendered possible by the physical properties of S(35).     

Preparation of Escherichia coli 70S ribosomes labeled with 35S

[35S]--70S ribosomes (150 Ci/mmol) were isolated from E. coli MRE-600 cells grown on glucose-mineral media in the presence of [35S] ammonium sulfate. The labeled 30S and 50S subunits were obtained from [35S] ribosomes by centrifugation in a sucrose density gradient of 10--30% under dissociating conditions (0.5 mM Mg2+). The activity of [35S]--70S ribosomes obtained by reassociation of the labeled subunits during poly(U)-dependent diphenylalanine synthesis was not less than 70%. The activity of [35S]--70S ribosomes during poly(U)-directed polyphenylalanine synthesis was nearly the same as that of the standard preparation of unlabeled ribosomes. The 23S, 16S and 5S RNAs isolated from labeled ribosomes as total rRNA contained no detectable amounts of their fragments as revealed by polyacrylamide gel electrophoresis. The [35S] ribosomal proteins isolated from labeled ribosomes were analyzed by two-dimensional gel electrophoresis. The [35S] label was found in all proteins, with the exception of L20, L24 and L33 which did not contain methionine or cysteine residues.     

AUTORADIOGRAPHIC DETERMINATION OF S35 IN TISSUES AFTER INJECTION OF METHIONINE-S35 AND SODIUM SULFATE-S35

The loss of "bound" S³⁵ that occurs during various mounting procedures used in autoradiography was studied in healing surface wounds of rats treated with either methionine-S³⁵ or Na₂S³⁵O₄. Valid autoradiography of bound S³⁵ in this tissue is not possible until 48 hours after radiosulfate and 24 hours after radiomethionine injection, when the S³⁵ is almost entirely bound in large protein and polysaccharide molecules. Autoradiograms of S³⁵ given in both the organic and inorganic form reveal substantial over-all loss of the bound isotope from sections subjected to contact with solvents prior to autoradiography. A comparison of autoradiograms prepared by dry-mounting sections of frozen-dried tissue with autoradiograms of wet-mounted sections of the same tissue suggest that the loss is proportional to the extent of the contact with solvents. Evidence suggests that loss of the isotope occurs during contact of the ribbon or section itself with solutions after fixation and cutting and prior to radiation exposure. No appreciable loss of the bound isotope seems to occur during contact of the intact tissue specimen with a variety of fluid fixatives except for a marginal zone at the excision edges of the tissue. The potential hazard of displacement of the isotope during fixation, however, remains. Technics which prevent loss of the isotope and fogging of the nuclear emulsion permit the use of thinner sections and emulsion films and the fine resolution of image rendered possible by the physical properties of S³⁵.     

Evaluation in tobacco of the organ specificity and strength of therolD promoter,domain A of the 35S promoter and the 35S2 promoter

In order to study the expression in plants of therolD promoter ofAgrobacterium rhizogenes, we have constructed chimaeric genes placing the coding region of thegusA (uidA) marker gene under control of tworolD promoter fragments of different length. Similar results were obtained with both genes. Expression studies were carried out in transformed R₁ progeny plants. In mature transformed tobacco plants, therolD-gus genes were expressed strongly in roots, and to much lower levels in stems and leaves. This pattern of expression was transmitted to progeny, though the ratio of the level of expression in roots relative to that in leaves was much lower in young seedlings. The degree of root specificity inrolD-gus transformants was less than that of a gene constructed with domain A of the CaMV 35S promoter,domA-gus, but the level of root expression was much higher than with the latter gene. However, the level of expression of therolD-gus genes was less than that of agus gene with a 35S promoter with doubled domain B, 35S²-gus. TherolD-gus genes had a distinctive pattern of expression in roots, compared to that of the two other genes, with the strongest GUS activity observed in the root elongation zone and in vascular tissue, and much less in the root apex.     

90S pre-ribosomes include the 35S pre-rRNA,the U3 snoRNP,and 40S subunit processing factors but predominantly lack 60S synthesis factors

We report the characterization of early pre-ribosomal particles. Twelve TAP-tagged components each showed nucleolar localization, sedimented at approximately 90S on sucrose gradients, and coprecipitated both the 35S pre-rRNA and the U3 snoRNA. Thirty-five non-ribosomal proteins were coprecipitated, including proteins associated with U3 (Nop56p, Nop58p, Sof1p, Rrp9, Dhr1p, Imp3p, Imp4p, and Mpp10p) and other factors required for 18S rRNA synthesis (Nop14p, Bms1p, and Krr1p). Mutations in components of the 90S pre-ribosomes impaired 40S subunit assembly and export. Strikingly, few components of recently characterized pre-60S ribosomes were identified in the 90S pre-ribosomes. We conclude that the 40S synthesis machinery predominately associates with the 35S pre-rRNA factors, whereas factors required for 60S subunit synthesis largely bind later, showing an unexpected dichotomy in binding.     

Labeling of proteins with [35S]methionine and/or [35S]cysteine in the absence of cells

Incubation of [35S]methionine and [35S]cysteine with bovine albumin, globulin, catalase, hemoglobin, or human globulin resulted in incorporation of the 35S label into each of these proteins. Trichloroacetic acid (TCA) precipitation revealed that the percentage of label incorporated ranged from 1 to 15%. The 35S labeling was resistant to dissociation by reducing SDS-PAGE, prolonged dialysis against 4 M urea, heating, TCA precipitation, and dilution by gel filtration. The labeling effect was more efficient with [35S]cysteine than [35S]methionine. Incubation of 35S label with proteins differing in methionine and cysteine content revealed no requirement for sulfur-containing amino acids in the target protein. Protein carboxymethylation reduced but did not prevent 35S label incorporation. Amino acid analysis of labeled proteins revealed that the radioactive label was not consistently associated with an individual amino acid. Differences in the ability of various proteins to spontaneously label with these amino acids suggest caution in the interpretation of metabolic labeling experiments and the necessity for inclusion of additional controls. Alternatively, our experience indicates a potentially useful method for labeling proteins in the absence of cells.