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K Scherrer

Publications and source records attributed to K Scherrer.

At least 109 records · Page 6Linked to original sources
PubMed1981

Comparisons of proteins associated with duck-globin mRNA and its polyadenylated segment in polyribosomal and repressed free messenger ribonucleoprotein complexes.

EDTA dissociation of polyribosomes from duck erythroblasts allowed us to isolate the 15-S globin messenger ribonucleoproteins (mRNP) by sucrose gradient centrifugation or affinity chromatography on poly(U)-Sepharose or oligo(dT)-cellulose columns. Their protein composition was compared by one and two-dimensional electrophoresis in sodium dodecyl sulfate to the free 20-S mRNP containing the repressed fraction of globin mRNA [Vincent, A., Civelli, O., Maundrell, K., and Scherrer, K. (1980) Eur. J. Biochem. 112, 617--633]. The protein composition of the 15-S mRNP isolated by these methods in different ionic strength conditions, was characterized by a major 73 000-Mr polypeptide and seven minor polypeptides with Mr ranging from 45 000 to 68 000, all of which are slightly basic, and about five acidic ones in the 80 000--130 000-Mr range. All these are retained in the 15-S mRNP core particle isolated at 0.5 M KCl. At low ionic strength, in addition, a specific group of acidic polypeptides in the Mr range 35 000--105 000 was also found associated with globin mRNA. Oligo(dT)-cellulose chromatography of mRNP digested with ribonucleases A and T1 indicated that the 73 000-Mr major protein is bound to the poly(A) segment; some other proteins resolved as minor components interact with both the poly(A) and non-poly(A) regions of globin mRNA. Characterization of proteins interacting with the poly(A) segment of non-polyribosomal globin mRNA in 20-S free mRNP demonstrated the absence of the polyribosomal 73 000-Mr poly(A)-binding protein. Furthermore, it confirmed that the protein compositions of translatable polyribosomal and repressed free globin mRNP are very different. Indeed, the respective core (0.5 M KCl) particles contain only two possibly common polypeptides. The specificity of proteins associated with globin mRNA in two different functional states shown here supports the hypothesis of a role of mRNP proteins in translational control of mRNA.

Animals↗
PubMed1980

Ribonucleotide sequences non-adjacent to poly(A) participate in the poly(A)-protein complex in 15S duck globin mRNP particles.

The study of the interaction between mRNA and proteins in the polyribosomal 15 S duck globin messenger ribonucleoprotein complex showed that proteins protect specific mRNA sequences against digestion by the nonspecific micrococcal nuclease (Nucleic Acids Research 6 (8) 2787, 1979). Here we report the isolation of the poly(A)-protein RNP complex from nuclease digested 15 S mRNP by two different methods: sucrose gradient sedimentation and oligo(dT)-cellulose chromatography. We show by fingerprint analysis, that aprt from the periodically fragmented poly(A) segment, mRNA sequences adjacent and non-adjacent to the poly(A) segment are protected by the poly(A) binding proteins against nuclease digestion. The duck globin poly(A)-protein RNP complex, with a sedimentation coefficient between 7 S and 10 S, shows a characteristic protein composition, with a major 73,000 MW polypeptide and some minor components. The results are discussed in view of a dynamic ribonucleoprotein structure.

Animals↗
PubMed1980

Restriction mapping of cDNA recombinants including the adult chicken and duck globin messenger sequences: a comparative study.

A comparison of the organization of six avian adult globin messenger sequences is based on previously reported recombinant duck adult globin cDNA plasmids (Therwath et al., 1980) and the actual construction and characterization of pBR322 recombinant plasmids including the beta and the normal alpha A and alpha D chicken adult globin mRNA sequences. Identification of the cloned DNA was performed using hybridization-selection under conditions permitting complete purification in one step of the three globin mRNAs, and translation of the corresponding mRNA. Orientation of the globin insert in the vector was determined, taking into account the computer prediction of the restriction sites based on the known amino acid sequences of the three globin chains (Roizès and Pelaquier, 1980) and those actually observed, and by identification of restriction fragments using 3'-specific probes. Identification, orientation and restriction mapping of these cloned DNAs reveals extensive homologies in organisation of beta sequences between duck and chicken, as well as among the alpha sequences in every two possible combinations.

Animals↗
PubMed1980

Identification and characterization of the translationally repressed cytoplasmic globin messenger-ribonucleoprotein particles from duck erythroblasts.

Globin messenger ribonucleoprotein (mRNP) particles which have been isolated from duck erythroblast post-polyribosomal supernatant are translationally inactive in vivo and in vitro but contain translatable mRNA active after deproteinisation. They were characterized following purification by successive sucrose gradient sedimentation in a buffer containing 0.05 M KCl. The complex, which sediments homogeneously at about 20 S, has a density of 1.39 g/cm3 and thus consists of four parts protein to one part RNA; 40% of this RNA is globin mRNA and no other mRNA could be detected. Sedimentation of the purified globin mRNP on sucrose gradients in 0.5 M KCl produced four components while polyacrylamide gel electrophoresis in non-denaturing conditions and in the presence of EDTA resulted in the separation of three components. Hybridization to globin cDNA and translation in vitro of the RNA extracted from these subparticles revealed the existence of two core particles containing globin mRNA with nominal sedimentation coefficients of 13 S and 16 S. Analysis of the protein components of the isolated sub-complexes by dodecyl sulfate and bidimensional gel electrophoresis indicated a very characteristic protein composition for each of these complexes. The 16-S and 13-S globin mRNPs differed essentially by the presence in the 13-S mRNP only of a group of major polypeptides. Of the other two sub-complexes, one consisted of 90% small RNA in the 4-S range; the second sedimented ahead of the globin mRNP core particles at about 19S and consisted of a very characteristic set of about 14 polypeptides. The polyribosomal 73000-Mr poly(A)-binding protein was not detected in the purified free globin mRNP although the mRNA in the untranslatable particle is polyadenylated. The presence in the cytoplasm of duck erythroblasts of two forms of untranslated globin messenger ribonucleoprotein particles, distinct in their protein composition from polyribosomal globin mRNP, suggests that they may have a specific role in the regulation of translation of globin mRNA.

Animals↗
PubMed1979

A rapid and sensitive method for detection of proteins in polyacrylamide SDS gels: staining with ethidium bromide.

We describe here a fluorometric method of detection of proteins fractionated by electrophoresis in polyacrylamide-SDS gels. This method, using ethidium bromide as fluorescent dye, is performed within 40 minutes after the end of the electrophoretic run. It does not require treatment of proteins prior to electrophoresis, and entails neither fixation of proteins in the gel, nor destaining. It is sufficiently sensitive to detect 0.5 - 1.0 microgram of protein per band. Furthermore, the simultaneous electrophoretic resolution and detection of protein and RNA on a single SDS-polyacrylamide gradient gel is reported.

Electrophoresis, Polyacrylamide Gel↗
PubMed1979

Evidence for the protection of specific RNA sequences in globin messenger ribonucleoprotein particles.

Purified 15 S globin mRNA-protein (mRNP) complexes obtained by EDTA dissociation of duck reticulocytes polyribosomes were digested with the calcium dependant Staphylococcus aureus nuclease (EC 3. 1. 4. 7.). 25% of the globin mRNA sequences were resistant to extensive nuclease digestion as determined by TCA precipitation of the digested 15 S particles labelled in vivo with tritiated uridine. Polyacrylamide gel electrophoresis of the RNA from nuclease digested 15 S particles showed that the protected oligoribonucleotides were distributed into two distinct size classes of 25,000 and 12,000 MW. Comparison between in vitro iodine-labelled 9 S globin mRNA extracted from Staphylococcal nuclease digested 15 S mRNP particles was carried out by fingerprinting. Mapping of T1 ribonuclease digests by high-voltage electrophoresis and homochromatography showed that specific oligoribonucleotides were protected against nuclease attack by proteins of the 15 S mRNP.

Animals↗
PubMed1979

On pre-messenger RNA and transcriptions. A review.

From the present review integrating old and new data emerge a few principles of gene expression in eukaryotes, and an infinite variety of possible mechanistic details generating the overal pattern. The few principles, most of which are not fundamentally new, may thus be summarized. 1) The eukaryotic genome is subdivided into transcriptional units: into transcriptons which are subject to individual activation controlled at DNA level. 2) Viral genomes may contain one or a few transcriptons, while cells of multicellular organisms contain from 3 x 10(3) in diptera up to an estimated 2 x 10(5) in birds and mammals. 3) Transcriptons may include one or several coding sequences. 4) Transcriptons vary considerably in size: in mammals and birds their size spectrum falls into the 2,000 to 20,000 bp range. 5) Units of coding information constituting one message (genes) and, possibly, units of regulative information are frequently broken up and stored within the transcripton in sub-genic blocks (of so far unknown significance) in general located at a certain distance from the 5' and 3' transcript terminals which are determined by the promotor and terminator signals. 6) The gene, in its specific definition as the functional unit underlying the phenotype, is in general constituted posttranscriptionally by the processing mechanisms from the mosaic of its genomic subunits in the transcripton; segments of coding, service and regulative sequences are recombined within themselves and with each other, polygenic transcripts separate into their unit messages. 7) Activated transcriptons produce pre-mRNA; these primary transcripts are colinear with the DNA of the transcriptional unit. 8) Primary pre-mRNA is processed into secondary pre-mRNA's by extragenic cleavage and intragenic ("splicing") processing, giving rise stepwise to functional mRNA. During this process chemical modifications as methylation, 5'-terminal capping and 3'-terminal polyadenylation take place. 9) Translation yields either potentially functional polypeptides or polycistronic polyproteins subject to further processing. 10) Processing is a regulated process; it involves many of the possible phases and mechanisms of post-transcriptional regulation (cf. 39, 40).

Animals↗
PubMed1979

Synthesis of albumin and malic enzyme in wheat-germ lysates and Xenopus laevis oocytes programmed with chicken-liver messenger RNA.

Undegraded, biologically-active, polyadenylated RNA was isolated from chicken liver by a rapid, simple procedure. Liver cells were dispersed mechanically and then broken gently by controlled Dounce homogenization in the absence of detergent or ribonuclease inhibitors. After removing lysosomes and mitochondria by centrifugation, RNA was precipitated at pH 5.2. Polyadenylated mRNA was isolated directly from the detergent-solubilized precipitate by oligo(dT)-cellulose chromatography. The resulting RNA was translated into liver-specific peptides in both the wheat germ lysate and Xenopus laevis oocytes. Translatable albumin mRNA was detected in the liver cytoplasm of both fed 3-week-old chicks and unfed, day-old chicks. Translatable malic enzyme mRNA was only detected in the livers from the fed chicks.

Albumins↗
PubMed1979

Characterization of pre-messenger-RNA-containing nuclear ribonucleoprotein particles from avian erythroblasts.

Ribonucleoprotein particles have been isolated from duck erythroblast nuclei using a procedure designed to produce maximal cytoplasmic dispersion with minimal release of endogenous hydrolytic enzymes. The RNA extracted from the purified nuclear ribonucleoprotein fraction is shown to contain globin messenger RNA sequences at a concentration comparable to that present in total nuclear RNA. The polypeptide composition of this fraction revealed by electrophoresis in two dimensions is complex, consisting of at least 65 acidic species and 21 basic species. Several lines of evidence suggest that these are authentic components of nuclear ribonucleoprotein. The so-called 'core' proteins of nuclear ribonucleoprotein which were previously shown to migrate as a single band on low-pH urea gels, and as six bands on sodium dodecyl sulphate gels are here shown to be considerably more complex being resolved by two-dimensional electrophoresis into a group of 15 basic and 6 more and less neutral polypeptides. Isoelectric focusing of nuclear ribonucleoprotein under non-denaturing conditions suggests that these latter species are not uniformly distributed along the pre-messenger RNA molecule.

Amino Acids↗
PubMed1978

Preparative density gradient centrifugation of RNA and DNA in cesium sulfate-urea mixture.

A new procedure is described for a large scale separation and purification of unfixed DNA and RNA from a mixture of partially extracted nucleic acids and lysates of subcellular fractions by centrifugation to equilibrium in cesium sulfate-urea mixture. Optimum conditions are described for the separation and quantative recovery of both RNA and DNA in a pure form. The procedure allows determination of peak buoyant densities of 4-5s RNA, 7-11s mRNA and total cytoplasmic RNA. The procedure also allows fractionation of small molecular weight classes of cytoplasmic RNAs from the 18s and 28s rRNAs.

Animals↗
PubMed1978

Post-transcriptional suppression of globin gene expression in cells transformed by avian erythroblastosis virus.

Cells transformed by avian erythroblastosis virus were grown in vitro for up to 5 months. After a few days in culture, synthesis of hemoglobin was undetectable and could not be induced by dimethyl sulfoxide. As shown by globin cDNA hybridization to nuclear and cytoplasmic RNA carried to Crot values of 10(5) moles of nucleotide per liter X sec, globin genes in these cells are transcribed into pre-mRNA, but no trace of globin mRNA appears in the cytoplasm. The implications of this observation for schemes of post-transcriptional regulations and viral transformation are discussed.

Alpharetrovirus↗