Biomedical subjects
Publications and source records attributed to C Coutelle.
Human globin cDNA-derived recombinants with plasmid pCR1 have been prepared for use as specific hybridisation probes and for the partial sequencing of alpha-, beta- and gamma-globin genes.
The cloning of DNA sequences in plasmid recombinants has made it possible to amplify specific sequences to an extent that they can be used for preparative purposes. We describe the use of rabbit globin DNA sequences cloned in the plasmid pCR1 and covalently bound to Sepharose 4B for the purification of chain-specific rabbit alpha- and beta-globin cDNAs. These purified probes were then used to estimate the length of the alpha- and beta-globin DNA sequences inserted into the recombinant plasmid. The technique should allow the rapid isolation of sequence-specific cDNA, RNA and genomic DNA.
Precursor mRNA (pre-mRNA) was extracted from erythroid enriched bone marrow cells of the rabbit by the methods of Georgiev and Mantieva modified by Markov and Arion and of Holmes and Bonner, respectively. Density gradient centrifugation, base analysis and the effects of alpha-amanitin and actinomycin D on the synthesis of the cellular RNA showed signs of degradation in the rRNA-free 85 degrees C-fraction of the preparation according to Georgiev and Mantieva and a substantial rRNA contamination of the 65 degrees C-fraction. This RNA-fraction as well as the total RNA-preparation extracted according to Holmes and Bonner was purified from rRNA by affinity chromatography on poly(U)-Sepharose. Poly(A)+-RNA of all size-classes, among it a substantial amount of high molecular weight RNA (greater than 45 S), was isolated by this purification procedure. Especially the extraction according to Holmes and Bonner yields high molecular weight material but the critical step of this procedure often resulting in degradation of the RNA is the DNase treatment of the heavily DNA-contaminated total RNA-preparation either due to RNase contamination of the DNase or to the existence of RNase in the less intensive deproteinized RNA. The investigated cellular system is characterized by a very intensive rRNA synthesis which is typical for cells in the early stages of hematopoiesis. In contrast to investigations with purified RNA-polymerases and subcellular systems, but in accordance with data of in vivo experiments, alpha-amanitin inhibits both the pre-mRNA and the pre-rRNA synthesis.
Pre-mRNA from bone marrow of rabbits enriched in erythroid cells was analyzed by T1 and pancreatic RNase treatment and poly(U)- and poly(A)-Sepharose chromatography to contain poly(A)-, oligo(U)- and double stranded sequences. The length of the poly(A)- and oligo(U)-sequences was determined by polyacrylamide gel electrophoresis using poly(A)- and oligo(U)-standards of defined length. Poly(A) from poly(A)+pre-mRNA isolated according to the method of Holmes and Bonner shows a size distribution between 40 and 130 nucleotides with an average of 75 nucleotides. Hot phenol extraction according to Georgiev et al. leads to a smaller size of about 25 nucleotides. The oligo(U)-segment consists of 80% U and is about 25 nucleotides long. Poly(A)+ pre-mRNA of about 12000--16000 nucleotides posseses 1--2 oligo(U)-units and one double strand of about 70 nucleotide pairs. Most (greater than 90%) of the oligo(U)-and the double stranded sequences are localized at least 1700 nucleotides away from the 3'terminus. Double strands were investigated with respect to their reannealing behaviour. The material consists of two types of double strands: 20% which reassociate at a cot/2 cot/2 of 1.3 . 10(-4) represent only one or a few types of double strands, the remaining 80% reassociate at a cot/2 of about 7 . 10(-2) and are more complex. Under hybridization conditions pre-mRNA molecules are able to self-annealation. 10% of the sequences become RNase stable.
Pre-mRNA fractions (greater than 45 S) were characterized by electron microscopy. High salt concentrations (0.2 M ammonium acetate, pH 8) yield linear molecules of different length (0.5--17 micrometer). In 10% of the molecules a compact-nonlinear contour (cn-contour) is detectable at one end. A significant enhancement of the number of cn-contour carrying molecules is observed after binding pre-mRNA to poly(U)-sepharose. The terminal cn-contour could be the depiction of a secondary and/or tertiary structure including the poly(A)-tail. 9 S globin mRNA appear in 80% with virtually the same cn-contour as detected in pre-mRNA molecules. After denaturing the mRNA in 80% formamide--4M urea in connection with heating to 90 degrees C from 10 min, a percentage of 77% of stretched, linear molecules results. This structural transformation is reversible when the denatured RNA is precipitated and redissolved in 0.2 M ammonium acetate. 73% of the stretched molecules are characterized by a mean length of 0.44 micrometer. This value is twice as high as commonly assumed for a globin mRNA chain.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
The cryoprotective effect of dimethylsulfoxide, glycerol and polyethylenglycol during freezing and thawing of human bone marrow was investigated by eosin staining test, an acridinorange fluochrome staining test and by RNA- and DNA-synthesis tests. In these tests the overall yield of vital nucleated cells, referred to the number in the absence of cryoprotectants and freezing and thawing, amounted to 50% with dimethylsulfoxide, 30% with glycerol, and 10% with polyethylenglycol. With dimethylsulfoxide and glycerol the loss of vital nucleated cells is almost entirely due to the addition of cryoprotectants. Polyethylenglycol freezing and thawing also leads to a great loss of vital nucleated cells. The results with dimethylsulfoxide show that the currently employed techniques of punction, preparation, freezing and thawing of bone marrow are suitable for clinical application.
Globin mRNA isolated from rabbit reticulocytes showed an unusually high molecular weight for the beta-chain mRNA (280000 daltons), a large difference between the molecular weight of the alpha-chain mRNA (230000 daltons) and the beta-chain mRNA and a dense and compact structure in the electron microscope (average length of the molecules 0.14 micron), which can be transformed to a linear structure (average length of the molecules 0.44 micron corresponding to a molecular weight of about 420000 daltons). The poly(A)-tail of the mRNA estimated by polyacrylamide gel electrophoresis with poly(A)-markers as reference substances has a size between 12 and 52 nucleotides. The mRNA works as a good template in the cDNA-synthesis with reverse transcriptase and full length copies of the mRNA were obtained by this reaction. The cDNA hybridizes to an amount of 70% with the globin mRNA at a 1:4 cDNA/mRNA ratio.
A test for the cellular RNA-synthesis (incorporation of 3H-uridine in the RNA) of human bone marrow has been standardized with respect to the time of incorporation, the number of cells and the concentration of 3H-uridine. The following parameters were estimated for 500 microleter standard assay and 100 microleter aliquots for the determination of the radioactivity: time of incubation 80 min, number of nucleated cells 8 - 10(5), concentration of 3H-uridine 8,3 - 10(-6) M. Actinomycin D inhibits the RNA-synthesis to 90% in a concentration of 1.2 - 10(2) microgram/ml. The test appears generally applicable for the determination of the vitality of bone marrow after cryopreservation, the testing of cryoprotectants and haematotoxic substances and the control of the reaction of the bone marrow during chemical- or irradiation treatment of tumors.
Melted ds RNA isolated from rabbit bone marrow pre-mRNA was hybridized with excess of globin mRNA which was prepared from rabbit reticulocytes. 7-9% of ds sequences became RNAase-stable and about 30% of the sequences could be bound to poly(U)-Sepharose through poly (A) of mRNA. The size of RNAase-stable hybrid is about 30 nucleotides, that is one fourth of the length of one strand of the ds RNA.
A system for the measurement of the RNA-synthesis of bone marrow cells of the rat has been developed and the incorporation of [3H]-uridine into the cellular RNA has been standardized with respect to the time of incubation, the concentration of [3H]-uridine and the number of cells. A plateau of the incorporation of [3H]-uridine into the RNA is reached after 20 min of incubation and is interpreted as the expression of a steady state in synthesis and degradation of the cellular RNA. A constant labelling of the RNA is reached above 8.3 with 10(-6)M [3H]-uridine. The optimal cell number in the 500 mul standard assay is 4 with 10(6). Actinomycin D inhibits the RNA-synthesis to 94% in a concentration of 1.2 with 10(2) mug/ml. The cryoprotectants dimethylsulfoxide, polyethylene-oxide and glycerol and the potential haematotoxic substances dichlorodiphenyltrichloroethane and gamma-hexane were tested in this system. 5% dimethylsulfoxide and 10% polyethylen-oxide in Eagle's-medium with ethylendiamintetra-acetate do not influence the RNA-synthesis. 5% glycerol reduces the incorporation of [3H]-uridine into the cellular RNA to about 30%.
Precursor mRNA (pre-mRNA) molecules, sedimenting at greater than 45 S, from erythroid-enriched bone marrow cells of the rabbit and hemoglobin mRNA molecules from rabbit reticulocytes were investigated by electron microscopy. Four of 98 measured pre-mRNA molecules had a length between 15 and 17.1 mum. In some of the pre-mRNA molecules a characteristic condensed structure was observed at one end, strikingly resembling the structure of the mRNA molecules.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.