5′-terminal caps of snRNAs are reactive with antibodies specific for 2,2,7-trimethylguanosine in whole cells and nuclear matrices : Double-label immunofluorescent studies with anti-m3G antibodies and with anti-RNP and anti-Sm autoantibodies

Antibodies specific for 2,2,7-trimethylguanosine (m3G), which do not cross-react with m7G-capped RNA molecules were used to study, by immunofluorescence microscopy, the reactivity of the m3G-containing cap structures of the snRNAs U1 to U5 in situ. In interphase cells, immunofluorescent sites were restricted to the nucleus, whilst nucleoli were free of fluorescence. This indicates that the 5' terminal of most of the nucleoplasmic snRNAs are not protected by an m3G cap-recognizing protein and that the snRNA caps are not necessarily required for the binding of snRNPs to subnuclear structures. The snRNAs in the nucleoplasm appeared as distinct units in the light microscope, and this allowed the comparison of the distribution of snRNP proteins by double label studies with anti-RNP or anti-Sm antibodies within the same cell. The three antibody classes produced superimposable fluorescent patterns. Taking into account that the various IgGs react with antigenic sites on snRNAs or snRNP proteins not shared by all the snRNP species, these data suggest that U1 snRNP particles are distributed in the same way as the other snRNPs in the nucleus. Qualitatively the same results were obtained with DNase-treated nuclear matrices indicating that intact snRNPs are part of the nuclear matrix. Our data are consistent with proposals that the various snRNPs may be involved in processing of hnRNA and that this may take place at the nuclear matrix.     

Mutants of Escherichia coli lacking ribosomal protein L1

Two independently isolated mutants of Escherichia coli, RD19 and MV17-10, that appeared to lack protein L1 on their ribosomes, as determined by two-dimensional gels, were subjected to a battery of immunological tests to find if L1 was indeed lacking. The tests involved Ouchterlony double diffusion, modified immunoelectrophoresis, dimer formation on sucrose gradients, and affinity chromatography. By all these criteria, protein L1 was missing from the ribosome in these mutants. Nor was any L1 cross-reacting material detectable in the supernatant. There was, however, a specific two- to fivefold increase in concentrations of protein L11 in the supernatants of the mutants, which was evidence that protein L1 acts as a feedback inhibitor of expression of the operon coding for the genes for proteins L11 and L1.Electron micrographs of ribosomes obtained from these mutants were indistinguishable from those of wild-type strains. 50 S ribosomal subunits from mutants RD19 and MV17-10 were reconstituted with purified L1 from wild-type and investigated by immunoelectron microscopy. The three-dimensional location of ribosomal protein L1 on the surface of the large subunit was determined. L1 is located on the wider lateral protuberance of the 50 S subunit. The position of protein L1 in 50 S subunits reconstituted from mutants RD19 and MV17-10 was indistinguishable from the position in subunits from wild-type.     

Localization and structure of snRNPs during mitosis. Immunofluorescent and biochemical studies

The distribution of U snRNAs during mitosis was studied by indirect immunofluorescence microscopy with snRNA cap-specific anti-m3G antibodies. Whereas the snRNAs are strictly nuclear at late prophase, they become distributed in the cell plasm at metaphase and anaphase. They re-enter the newly formed nuclei of the two daughter cells at early telophase, producing speckled nuclear fluorescent patterns typical of interphase cells. While the snRNAs become concentrated at the rim of the condensing chromosomes and at interchromosomal regions at late prophase, essentially no association of the snRNAs was observed with the condensed chromosomes during metaphase and anaphase. Independent immunofluorescent studies with anti-(U1)RNP autoantibodies, which react specifically with proteins unique to the U1 snRNP species, showed the same distribution of snRNP antigens during mitosis as was observed with the snRNA-specific anti-m3G antibody. Immunoprecipitation studies with anti-(U1)RNP and anti-Sm autoantibodies, as well as protein analysis of snRNPs isolated from extracts of mitotic cells, demonstrate that the snRNAs remain associated in a specific manner with the same set of proteins during interphase and mitosis. The concept that the overall structure of the snRNPs is maintained during mitosis also applies to the coexistence of the snRNAs U4 and U6 in a single ribonucleoprotein complex. Particle sedimentation studies in sucrose gradients reveal that most of the snRNPs present in sonicates of mitotic cells do not sediment as free RNP particles, but remain associated with high molecular weight (HMW) structures other than chromatin, most probably with hnRNA/RNP.     

Growth-dependent regulation in production and utilization of acetylated ribosomal protein L7.

The relative levels of protein L12 and its α-N-acetylated form L7 in ribosomes of Escherichia coli have previously been shown to markedly vary during the growth cycle. The present labeling study shows preferential utilization of L12 in early logarithmic phase and of L7 in late logarithmic phase. Both forms are, however, simultaneously used throughout the growth cycle. After assembly into ribosomes, L7 and L12 are conserved without net interconversion. It is therefore concluded that the variation in L12 to L7 ratio takes place through changes in the relative flow of L7 and L12 species into ribosome assembly rather than by modification in pre-existing ribosomes. During this study, we have also measured the surprisingly large difference in the binding of Coomassie Blue to these proteins.     

Selective separation procedure for determination of ribosomal proteins L7 and L12.

An electrophoretic procedure for the selective separation and determination of the closely similar ribosomal proteins L7 and L12 (which are specifically involved in the GTPase reactions of the ribosome) from the total protein mixture extracted from unwashed ribosomes is described. In this procedure, which takes advantage of their unusually low isoelectric points. L7 and L12 (and a few other acidic proteins) migrate into gel asanions, while the bulk of ribosomal proteins which are basic remain behind. The positions of L7 and L12 were determined with authentic, pure proteins. It was further determined by means of 2-dimensional gel electrophoresis that no other protein components present in unwashed ribosomes comigrate with the bands of L7 and L12.     

Structural dynamics of bacterial ribosomes: II. Preparation and characterization of ribosomes and subunits active in the translation of natural messenger RNA

Ribosomes from Escherichia coli were tested for activity in initiation with R17 RNA as messenger. All vacant 70 S ribosomes but not all subunits were found to be active. The ability of 30 S and 50 S subunits to form a 70 S couple at Mg²⁺ concentrations above 4 mm is a stringent test for activity.Fresh extracts, prepared at 10 mm-Mg²⁺ from cells harvested after slow cooling contain up to 80% of the ribosomes in the form of vacant 70 S couples and 20% of free subunits. The proportion of subunits increases with standing as a result of the preferential inactivation of the 50 S particles. “Native” subunits are heterogeneous and consist mostly of active 30 S and inactive 50 S particles.In contrast to 50 S subunits, 30 S subunits prepared by exposure of 70 S ribosomes to low Mg²⁺ concentrations, are largely inactive and unable to reassociate with their active 50 S counterparts. However, both initiation and association activity can be restored by heating.The results imply that the structures necessary for subunit association are most critical for the biological activity of ribosomes, presumably because they are topologically closely related to the binding sites for messenger RNA, transfer RNA, and the protein factors for initiation, translocation and termination.     

An improved method for the separation of peptides and alpha-amino acids on copper-Sephadex.

An improved method for the separation of peptides from large amounts of α-amino acids on copper-Sephadex is described. The separation is essentially dependent upon the copper content of Sephadex, the pH of the system, and the concentration of the sample, and it is due to the different stabilities of copper complexes of Sephadex, peptides, and α-amino acids. Sephadex behaves as a solid ligand. Compounds that form weaker complexes with copper than Sephadex apparently move with the solvent front. α-Amino acids that form copper complexes having a stability comparable to that of copper-Sephadex complexes are retained on the column. Peptides form strong complexes, stripping copper from the column. They are only slightly retained. The method is most practical when copper is removed from the eluates with chelating ion-exchanger Dowex A-1. Examples are given for the chromatography of single compounds, model mixtures, and extracts from cheese and yeast.     

Cell patterning in branched and unbranched fruiting bodies of the cellular slime mold Polysphondylium pallidum

Cell patterning, the percentage of spores and stalk cells, was measured in branched and unbranched asexual fruiting bodies of Polysphondylium pallidum. Unlike D. discoideum, where small and large fruiting bodies are more stalky than average-sized fruiting bodies, the overall cell patterning was the same in branched and unbranched fruiting bodies of all sizes in P. pallidum. Light greatly increased the numbers of fruiting bodies in P. pallidum per unit area (or decreased aggregation territory size) so that most fruiting bodies formed in the light were small and unbranched. By contrast, light had little effect on the cell patterning of P. pallidum, although there was a slight increase in the percentage of stalk cells in the light compared to the dark. This indicates that the mechanisms governing light sensitivity of aggregation territory size and cell patterning have different components in P. pallidum. The accuracy of cell patterning of individual branches of branched fruiting bodies was so imprecise as to leave doubt that patterning is occurring at the branch level. Individual whorls of branched fruiting bodies had a greater percentage spores (90%) than whole fruiting bodies (78%) and the cell patterning was relatively imprecise. Only in whole fruiting bodies was the spore:stalk ratio highly correlated. These findings are consistent with cell pattern determination operating at the whole aggregate level, rather than at the individual whorl or branch level in P. pallidum.     

An improved method for preparing large arrays of bacterial colonies containing plasmids for hybridization: In situ purification and stable binding of DNA on paper filters

An improved procedure is presented for the binding to filter paper and subsequent purification of DNA from plasmid-containing bacterial colonies. The procedure includes treatments with NaOH, enzymatic digestion, and organic solvent extraction of the filter-bound DNA. This method allows isolation of DNA in a reusable form from thousands of colonies in several hours. Double-labeling experiments with [³H]thymidine and [¹⁴C]proline indicated that (i) during purification the DNA:protein ratio is increased several hundredfold; (ii) little or no DNA is lost during the procedure; (iii) the resultant purified DNA is tenaciously bound to the paper. Thus, the final filter-bound DNA allows multiple sequential hybridizations of different probes to one filter.     

The murine bone marrow macrophage,a sensitive indicator cell for murine migration inhibitory factor and a new method for their harvest

Murine bone marrow macrophages grown on Teflon-coated petri dishes for a period of 8–16 days can be removed with a yield of 90–95% and a viability greater than 95% following incubation in 1 mM EDTA. Bone marrow cells cultured on Teflon-coated dishes did not differ in their replication rate, peroxidase and nonspecific esterase content, pinocytosis, secretion of lysozyme and neutral proteinases from bone marrow cells cultured on plastic dishes. Murine bone marrow macrophages were found to be sensitive indicator cells for mouse migration inhibitory factor (MIF). Large numbers of cells for the MIF assay can be obtained, since their yield is 10–15 times higher than the yield of oil-induced peritoneal exudate macrophages from the same number of mice.     

Ceramide and inositol content of the lipopeptidophosphoglycan from Trypanosoma cruzi.

Inositol (2.5%), 17-methyl-sphinganine (4.8%) and sphinganine (1.5%) have been identified as constituents of the lipopeptidophospholycan isolated from whole cells of epimastigote forms of $\text{Trypanosoma cruzi}$. The branched chain base was characterized by combined gas chromatography — mass spectrometry.     

Messenger-like RNA synthesis and DNA chromosomal puffs in the salivary glands of Rhynchosciara americana

RNA synthesis was studied mainly in the proximal sections of Rhynchosciara salivary glands in late fourth instar at two typical periods of development. These are characterized either by the absence or presence of the so-called “DNA puffs” in the salivary gland chromosomes. It was found that simultaneously with the appearance of the DNA puffs there is a great increase in the synthesis of all RNA species. The greatest increase was found to take place in the rate of synthesis of messenger-like RNA. Four main classes of messenger-like RNA were detected, having mobilities corresponding to 33, 23, 16, and 14 S RNA. There is a correlation between the abundance of the 16 S messenger-like RNA and the degree of opening of the B-2 DNA puff. This species might therefore be transcribed from this puff.     

A convenient large-scale method for the isolation of membrane vesicles permeable to a specific inorganic ion: Isolation and characterization of functional acetylcholine receptor-containing vesicles from the electric organ of Electrophorus electricus

A convenient, large-scale method for the isolation of membrane vesicles permeable to specific inorganic ions has been developed. The general principle of this method involves the exchange of Na⁺ within the vesicles for external Cs⁺. Vesicles in which this exchange rapidly occurs can be separated on the basis of their density from vesicles in which the exchange occurs slowly (G. P. Hess and J. P. Andrews (1977) Proc. Nat. Acad. Sci. USA74, 482–486). This approach has been adapted to develop a method suitable for the large-scale isolation of vesicles that contain functional acetylcholine receptors from the Electrophorus electricus electroplax. The new procedure involves a discontinuous sucrose gradient for an initial purification of the vescles. This allows the use of a low-speed centrifuge, which has a capacity up to 30 times greater than the Beckman ultracentrifuge previously used. A self-forming CsCl-Percoll gradient and low-speed centrifugation are then used for the isolation of the functional acetylcholine receptor-containing vesicles. The isolation step leads close to the theoretically possible fourfold purification of the vesicles that contain functional receptors. The yield, up to 12 mg membrane protein/centrifugal run, is about 100-fold higher than the yield from the sucrose-CsCl density gradient previously (Hess and Andrews, see above) used. The gradients are self-forming and an equilibrium is reached after centrifugation for only 30 min. In 12 experiments with membrane preparations from 12 different ceis, the functional vesicles had an internal volume of 2.0 ± 0.3 μl/mg vesicle protein and a receptor concentration of 1.2 ± 0.02 μm (1.2 μmol/liter of internal volume). Electron micrographs of these vesicles show an average vesicle radius of 1600 ± 300 Å. From these results, an average of 12 receptor molecules/membrane vesicle is calculated.     

A one-step procedure for the isolation of fructose 1,6-bisphosphatase and fructose 1,6-bisphosphate aldolase from rabbit liver

Human ceruloplasmin, which is usually cleaved by limited proteolysis into three major fragments during preparation ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{? 18,650}$, 50,000, and 70,000) was isolated in good yield as an undegraded single-chain protein ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{? 135,00}$). The cryosupernatant from fresh frozen plasma (100 liters) was fractionated with polyethylene glycol (PEG 4000) at + 5°C yielding a ceruloplasmin-enriched fraction in the 20% PEG supernatant. Three steps of chromatography on DEAE-Sephacel, hydroxyapatite, and Sephadex G-200 produced a homogeneous protein with maximal enzymatic activity and the $\text{A}{}_{610}\text{A}{}_{280}$ ratio of 0.046 corresponding to 98–100% purity. Two forms of ceruloplasmin having this absorbance ratio were obtained; Form I was predominant and was studied further. The procedure separated both forms from apoceruloplasmin and degraded ceruloplasmin. The single-chain ceruloplasmin (Form I) had an NH₂-terminal sequence of Lys-Glu-Lys-His-Tyr-Tyr-Ile-, the same as for the 70,000 fragment, and is suitable for structural study by sequence analysis and physicochemical methods.     

A method for visualizing and quantifying the total complement of free and membrane-bound ribosomes in rat liver.

A method is described for both visualization and quantification of the total complement of rat liver free and membrane-bound ribosomes, undegraded by nucleolysis and unaggregated by pelleting. The method involves: (a) differential centrifugation of liver homogenate which separates free and membrane-bound ribosomes; (b) treatment of the fractions with detergents to solubilize membranes and remove nuclei; (c) centrifugation of a portion of each fraction to remove all the ribosomes; (d) sedimentation of the samples and blanks on sucrose gradients; and (e) difference photometric scanning of the gradients, sample minus ribosome-free blank, to detect the ribosomes free of interference from nonribosomal materials. The use of the SW 56 rotor in the initial centrifugation and of a high Mg²⁺ concentration (20 mm) in the medium used to suspend the bound fraction prior to detergent treatment were found to be essential in obtaining bound polysomes of large size (～19-somes). The difference scanning technique is shown to be a sensitive, accurate, and reproducible means of eliminating interference from nonribosomal materials, principally detergents and protein, and of quantifying ribosomes in both fractions. The method is rapid (3.5 h), simple to perform, and well suited for the analysis of multiple liver samples. It can be used to assess the concentration, distribution, organization, and average size of the total complement of rat liver free and membrane-bound ribosomes in a single experiment.