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D Elson

Publications and source records attributed to D Elson.

At least 19 recordsLinked to original sources
PubMed1987

A three-dimensional model of domain III of the Escherichia coli small ribosomal subunit.

A three-dimensional model of domain III (nucleotides 920 to 1395) of the 30S ribosomal subunit of E. coli is proposed. The data used as a guide in folding the secondary structure of the RNA into a tertiary structure are four long range RNA-RNA interactions proposed by us on the basis of experiments performed in this laboratory plus two sets of data from other laboratories: protein-RNA cross-linking sites for proteins S1, S3, S7, S10 and S12, and the interprotein distances determined by neutron scattering. The model is consistent with nearly all of the published experimental findings on the structure of domain III.

Base Sequence↗
PubMed1985

Long range RNA-RNA interactions in the 30 S ribosomal subunit of E. coli.

We have attempted to identify long-range interactions in the tertiary structure of RNA in the E. coli 30 S ribosome. Native subunits were cleaved with ribonuclease and separated into nucleoprotein fragments which were deproteinized and fractionated into multi-oligonucleotide complexes under conditions intended to preserve RNA-RNA interactions. The final products were denatured by urea and heat and their constituent oligonucleotides resolved and sequenced. Many complexes contained complementary sequences known to be bound together in the RNA secondary structure, attesting to the validity of the technique. Other co-migrating oligonucleotides, not joined in the secondary structure, contained mutually complementary sequences in locations that allow base-pairing interaction without disrupting pre-existing secondary structure. In seven instances the complementary relationship was found to have been preserved during phylogenetic diversification.

Base Sequence↗
PubMed1985

Identification of the collar-like structure of the 30S ribosomal subunit from E. coli by dark field electron microscopy.

We have used dark field electron microscopy to study a fragment of the small (30S) subunit of the E. coli ribosome. This fragment is almost the same size as the parent particle but RNA sequencing studies have shown it to lack, as a major constituent, a 150-nucleotide stretch at the 3' end of the rRNA, and two minor sections constituting 20 nucleotides from the 5' end and the 15 nucleotides of the sequence 687-701. The protein composition of the fragment was essentially unchanged. Samples of this material, and controls, were examined in the electron microscope after treatment with a buffered uranyl acetate solution for positive staining. Careful comparison revealed the following differences. The structural feature that we call the "collar" was missing in the fragment. Of the three parallel uranyl-staining bands that we have observed in micrographs of whole 30S subunits, the fragment consistently lacked the uppermost band. These observations identify the top uranyl-adsorbing band as being the 3' end of the ribosomal RNA and show that it can be equated with the collar-like structure.

Base Sequence↗
PubMed1983

A survey of 50 S ribosomal subunits by dark field electron microscopy.

A survey of dark field electron micrographs of the 50 S ribosomal subunit of E. coli has been performed and supplemented, for comparative purposes, by examination of negatively stained or metal shadowed specimens in the bright field mode. Attention was directed to the so-called "crown" and "kidney" views. The elongated appendage seen in negatively stained crown profiles was not observed in unstained or positively stained samples examined in dark field; these showed only symmetrical crown profiles regardless of changes in buffer type and drying method and of the presence or absence of uranyl acetate treatment and glutaraldehyde fixation. The crown view occasionally displayed a bifurcation in one of the lateral lobes, while the kidney profile showed a groove near the base of the convex edge. Uranyl acetate treatment produced delicate stripes which may give an indication of the surface RNA distribution.

Escherichia coli↗
PubMed1983

Specific visualization of ribosomal RNA in the intact ribosome by electron spectroscopic imaging.

The recently developed electron microscopic technique of electron spectroscopic imaging has been used to map the distribution of phosphorus, and therefore of RNA, in situ in the ribosomal subunits of E. coli. The results indicate that the RNA moiety of both subunits is concentrated toward the centre of the particle somewhat more than is the total mass, but reaches the outer surface at several places. The micrographs also reveal certain distinctive features in the shape of the RNA component that may be related to the overall shape of the ribosome. The method yielded a reasonably accurate estimate of the phosphorus content of the 30 S ribosome.

Microscopy, Electron↗
PubMed1982

A ribonucleoprotein fragment of the 30 S ribosome of E. coli containing two contiguous domains of the 16 S RNA.

Ribonucleoprotein fragments of the 30 S ribosome of E. coli have been prepared by limited ribonuclease digestion and mild heating of the ribosome in a constant ionic environment. One such fragment has been described previously. A second electrophoretically homogeneous fragment has now been isolated and its RNA and protein moieties have been characterized. It contains the 5' half of the 16 S RNA, encompassing domains I and II except for the extreme 5' terminus and several small gaps. Seven proteins are present: S4, S5, S6, S8, S12, S15 and S20. The RNA binding sites of five of these proteins are known, and all are RNA sequences that are present in the fragment. Published neutron scattering and immuno-electron microscopic data indicate that six of the proteins are clustered together in a cross sectional slice through the center of the subunit. After deproteinization, the RNA moiety gives two bands in gel electrophoresis, one containing domains I and II and the other, essentially only domain II. The former, although larger, migrates faster in gel electrophoresis, indicating that RNA domains I and II interact with each other in such a way as to become more compact than domain II by itself.

Base Sequence↗
PubMed1982

The topology of the 30 S ribosomal subunit and a proposal for the surface distribution of its RNA by dark field electron microscopy.

A three-dimensional model of the Escherichia coli 30 S ribosome has been derived from an extensive dark field electron microscopic investigation of unstained and unshadowed ribosomes. Subsequent studies of specimens lightly stained with uranyl acetate have added information on the location of regions that preferentially bind uranyl ions and may represent surface RNA. The model is an elongated cone-shaped structure 180-200 A long and is divided into identifiable sections: a "head" partly encircled by a collar-like structure, a middle section carrying a 20-A protuberance, and a "tail." A groove separates the middle section from the tail. Uranyl-binding regions can be seen on the protuberance and the collar's ridge, in two bands below the collar and parallel to it, and in a patch on the back of the head. The staining technique described may offer a method for mapping the external nucleic acid of nucleoprotein complexes.

Escherichia coli↗
PubMed1982

A ribonucleoprotein fragment of the 30 S ribosome of E. coli: evidence for long range RNA-RNA interactions.

A stable homogeneous ribonucleoprotein fragment of the 30 S ribosomal subunit of E. coli has been prepared by mild nuclease digestion and heating in a constant ionic environment. The fragment contains about half of the 16 S ribosomal RNa and six proteins: S4, S7, S9, S13, S16 and S19. The RNA moiety contains the reported binding sites of all six proteins. After deproteinization, 80% of the RNA migrated as two major electrophoretic bands, which were isolated and sequenced. Each band contained sequences from the 5' and 3' thirds of the 16 S RNA but none from the central third. That these two noncontiguous RNA domains migrated together electrophoretically in Mg++-containing gels after deproteinization constitutes direct evidence that the 16 S RNA is folded in the intact ribosome so as to bring the two domains close together and that there are RNA-RNA interactions between them in the presence of Mg++.

Base Sequence↗
PubMed1982

Cytogenetic studies of peripheral myeloblasts and bone marrow fibroblasts in acute myelofibrosis.

Chromosome analysis with giemsa-trypsin banding was performed on circulating myeloblasts and cultured bone marrow fibroblasts from a patient with acute myelofibrosis. Major karyotypic abnormalities were found in the myeloblasts involving chromosomes #1, 3, 5, 13, and 16; no chromosome abnormalities were detected in the fibroblasts. In addition, a pericentric inversion of number 7 was present in the circulating blasts but not the fibroblasts. Pericentric inversions have not previously been associated with acquired abnormalities of malignant cells. These findings support the concept that acute myelofibrosis is a primary malignancy of hematopoietic cells associated with secondary marrow fibrosis. The entity of acute myelofibrosis is discussed and the results of cytogenetic studies of previous reports are reviewed.

Acute Disease↗
PubMed1981

Water soluble receptors for human growth hormone from rabbit liver.

Soluble receptors that bind human growth hormone have been prepared by incubation of liver membranes form pregnant female rabbits in 1 mM Tris buffer (ph 7.5 or 9.0) at 4 degrees C. Up to 29% of the growth hormone binding sites could be solubilized within 48 hours. The kinetics of binding of human growth hormone to the soluble receptor, the hormonal specificity and the binding parameters calculated by Scatchard analysis (Ka 2.2 x 10(9) M-1, capacity 409 fmole/mg) were essentially unchanged compared with those for the parent membrane-associated (particulate) receptor. Gel filtration on Ultrogel AcA22 indicated that the major binding peak eluted at a molecular weight of 300,000 daltons. Specificity studies showed that the soluble binding sites had a moderately high affinity for ovine prolactin (Ka integral of 1 x 10(8) M-1), but negligible affinity for insulin. Although aqueous extraction gives a lower yield of binding sites for human growth hormone than detergent extraction, it nevertheless avoids some of the problems associated with use of detergents and should facilitate the subsequent purification of the receptor in a relatively unaltered state. It may also have applicability for solubilization of other hormone receptor systems.

Animals↗
PubMed1979

Binding of magnesium ions and ethidium bromide: comparison of ribosomes and free ribosomal RNA.

Comparative studies of free ribosomal RNA and ribosomes were made with two probes, Mg++ ions and ethidium bromide, which interact with RNA in different ways. Mg++. E. coli 16 S rRNA and 30 S ribosomes were equilibrated with four different buffers. Equilibration required several days at 4 degrees and several hours at 37 degrees. In all buffers ribosomes bound more Mg than free rRNA, the difference sometimes reaching 20--30%. Ribosomes were more resistant than free rRNA to heat denaturation and their denaturation was more highly cooperative. Ribosomes that bound more Mg++ had higher denaturation temperatures. Ethidium bromide. Fluorescence enhancement studies of ethidium intercalation showed the free 16 S rRNA to have 50--80 binding sites per molecule. A large fraction of these sites were present and accessible in the ribosome, but their ethidium-binding constants were reduced by an order of magnitude. In addition, free rRNA contained a small number of very strong binding sites that were virtually absent in the ribosomes.

Chemical Phenomena↗
PubMed1979

A purified nucleoprotein fragment of the 30 S ribosomal subunit of Escherichia coli.

A '13 S' nucleoprotein fragment was isolated from a nuclease digest of Escherichia coli 30-S ribosomal subunits and purified to gel electrophoretic homogeneity. It contained two polynucleotides, of about 1.1 . 10(5) and 2.5 . 10(4) daltons, which separated when the fragment was deproteinized. The major protein components were S4, S7 and S9/11, with S15, S16, S18, S19 and S20 present in reduced amount.

Escherichia coli↗
PubMed1979

Interaction between the hepatic growth hormone receptor and concanavalin A.

The interaction between the plant lectin concanavalin A (Con A) and hepatic receptors for human growth hormone (GH) has been studied in particulate and soluble microsomal membrane preparations from rabbit and rat liver. Con A shows a dose-dependent, partial (30%) inhibition of 125I-human GH binding which is reversed by the Con A competitor, alpha-methyl mannoside. The Con A effect is dependent on the receptor concentration. The inhibition by Con A in rabbit liver is a reflection of a decreased number of available binding sites--there is no effect on binding affinity. It would appear that Con A binds directly to the GH-binding protein and not to an adjacent membrane glycoprotein. The GH receptor may consist of more than one molecular species, differing only in the carbohydrate type or content.

Animals↗
PubMed1978

A large nucleoprotein fragment of the 50-S ribosomal subunit of Escherichia coli.

A large nucleoprotein fragment was isolated from a nuclease digest of Escherichia coli 50-S ribosomes and purified to gel electrophoretic homogeneity. Conditions were employed under which the fragmentation pattern was reproducible and the various fragment fractions were stable and maintained their sedimentation and electrophoretic properties throughout the several preparative and analytical procedures used. Fractions that appeared homogeneous in sucrose gradient centrifugation were found to be heterogeneous by gel electrophoresis. The large fragment was purified to homogeneity by preparative gel electrophoresis. It contained 21 proteins, the 5-S RNA, and two large oligonucleotides which together total about two thirds the molecular weight of the 23-S RNA. Because it can be prepared reproducibly in large quantities and because of its size and stability, the fragment appears suitable for functional and structural studies and as the starting material for further fractionation. An important contributing factor to the observed stability and reproducibility was the maintenance of an unchanging ionic environment. A single buffer was employed throughout all the procedures, and the fragments produced by nuclease digestion were dissociated from each other by heat rather than by changing the medium.

Enzymes, Immobilized↗
PubMed1978

A photoaffinity labelling study of the messenger RNA-binding region of Escherichia coli ribosomes.

A photoaffinity labelling study of the messenger RNA-binding region of E. coli ribosomes has been made, using oligoadenylic acids as mRNA analogs. The oligonucleotides, of chain length 6 to 8 and thus several nucleotides longer than oligonucleotides previously employed for this purpose, carried a radioactive photolabile aromatic azide reagent bound covalently to the 3'-terminal ribose moiety. The synthesis of the reagent, p-azidobenzoyl-(3H)-glycylhydrazide, is described. The derivatized oligonucleotides were shown to be functional messengers. They stimulated the binding of the cognate aminoacyl-tRNA, lysyl-tRNA: their binding was reciprocally stimulated by lysyl-tRNA; and they competed with underivatized oligoadenylates for ribosomal binding sites. When the 70 S ribosomal binding complex was irradiated, the photolabile reagent reacted covalently with both RNA and proteins of the 30 S subunit and with tRNA, but not with the 50 S subunit. The 16 S RNA appeared to be labelled at more than one site. Of the proteins, S3 and S5 reacted with the reagent with high specificity; and the possibility was not eliminated that S4 may have been labelled to a minor degree. Functional studies in other laboratories have implicated S3 and S5 in the decoding process, but these proteins were not labelled by any of the previously reported mRNA affinity labelling analogs. The results reported here therefore indicate that S3 and S5 not only affect the decoding process, but are located in the mRNA-binding region of the ribosome, presumably to the 3' side of the decoding site.

Affinity Labels↗
PubMed1976

Conformational changes of 30S ribosomes measured by intrinsic and extrinsic fluorescence.

The intrinsic tryptophan fluorescence and the fluorescence of N-(3-pyrene)maleimide, a covalently bound sulfhydryl-specific extrinsic probe, have been used to study the conformation of the 30S ribosomal subunit of Escherichia coli. (a) The tryptophan fluorescenct spectrum of the free ribosomal proteins is shifted to shorter wavelengths than that of free tryptophan. When the proteins are incorporated into the organized structure of the ribosome, there is a small additional blue shift and the emission band becomes narrower. In 6 M urea, the spectrum of the proteins, whether free or in the ribosome, becomes identical with that of the amino acid, reflecting exposure of previously shielded tryptophan residues. (b) When magnesium-depleted ribosomes are unfolded at low ionic strength, the tryptophan fluorescence spectrum changes, although circular dichroism shows no change in alpha-helix content of the proteins. (c) Intrinsic and extrinsic fluorescence were both found to be sensitive to a limited and fully reversible transition that takes place when ribosomes are incubated under conditions that increase their activity in vitro. This suggests that both probes may be of use in monitoring conformational changes that occur under conditions consistent with activity. The kinetics of the concurrent changes in extrinsic fluorescence and aminoacyl-tRNA binding activity were compared. (d) Conditions are described for labeling ribosomes with N-(3-pyrene)maleimide without impairing their activity.

Circular Dichroism↗
PubMed1976

Synthesis of a cleavable protein-crosslinking reagent for the investigation of ribosome structure.

This communication describes a simple method for synthesizing cleavable bifunctional imido esters of different chain lengths. These reagents, which form covalent crosslinks between lysine residues of proteins, contain a disulfide bond which is cleaved under mild conditions by reducing agents such as 2-mercaptoethanol. The reagents are synthesized via the dithiobisnitrile which is prepared in high yield by reacting the appropriate omega-activated nitrile with sodium polysulfide and is then converted quantitatively to the diimidate. Three such reagents were prepared: dimethyl 3.3'-dithiobispropionimidate, dimethyl 4,4'-dithiobisbutyrimidate, and dimethyl 6-6'-dithiobiscaproimidate. The first was synthesized from acrylonitrile, and the others from the appropriate omega-bromonitriles. Experiments with the bispropionimidate and a test protein, pancreatic ribonuclease, have shown the reagent to be effective in producing multimeric crosslinked complexes, from which monomeric proteins can recovered after treatment with 2-mercaptoethanol. The reagents are suitable for studies of ribosomal structure.

Binding Sites↗