Biomedical subjects

G Spohr

Publications and source records attributed to G Spohr.

31 records · Page 2Linked to original sources

PubMed1980

Electron microscope study of duck globin mRNA precursor crosslinked in situ.

Double-stranded RNA segments present in duck globin pre-mRNA were crosslinked in situ with aminomethyltrioxalen and UV light. The secondary structure of the crosslinked pre-mRNA was then studied by electron-microscopic analysis of pre-mRNA . cDNA hybrids. The data suggest that duck globin pre-mRNAs contain intervening sequences that are excised stepwise. Excision and subsequent ligation appears to occur on precursor molecules that are stabilized by base-paired regions.

Animals↗

PubMed1979

Globin mRNA precursor. Cross-linking in situ of double-stranded segments with aminomethyltrioxalen.

The globin mRNA sequences present in duck erythroblast nuclei appear under non-denaturing conditions to be associated with heterogeneous nuclear RNA (hnRNA) molecules of various sizes. Under denaturing conditions, however, the bulk of the globin mRNA sequences associated with hnRNA are released as molecules of size close to that of the active globin mRNA. To find out whether hydrogen-bonded structures occur in situ or arise after RNA extraction, nuclei were treated with aminomethyltrioxalen and exposed to ultraviolet light. This treatment generates covalent links between opposite strands of double-stranded nuclei acids, which were visualised by electron microscopy. It appears that, after cross-linking, a fraction of the globin mRNA sequences present in nuclei is associated with high-molecular-weight hnRNA molecules by a link found associated with a band of 0.9 x 10(6) molecular weight approximately. It is suggested that within the erythroblast nucleus, globin mRNA sequences are associated by hydrogen bonds with RNA of high molecular weight. These structures may represent intermediate steps in globin mRNA processing.

Animals↗

PubMed1978

Messenger RNA synthesis in synchronized Chinese hamster ovary cells.

Chinese hamster ovary cells were synchronized without inhibitors by mitotic selection and labelled in G1, S or G2 phase by incubation for 90 min with [3H]- OR [14C]uridine. Purified polyribosomes were extracted with phenol and the polyadenylated mRNA prepared by poly(U)-Sepharose chromatography. Poly-adenylated [3H]uridine-labelled mRNA from the G1 phase of the cell cycle was compared by exponential polyacrylamide gel electrophoresis in formamide with [14C] uridine-labelled polyadenylated nRNA from the S or G2 phase. The electrophoretic patterns obtained correspond to the size range expected for mRNA (7-28 S). No prominent differences were detected between mRNAs synthesized in different phases of the cell cycle. From these data we conclude that the major size classes of polyribosomal poly(A)-containing mRNA are synthesized in equal ratios throughout the cell cycle.

Cell Cycle↗

PubMed1978

Kinetics of synthesis of cytoplasmic messenger-like RNA not associated with ribosomes in HeLa cells.

The turn-over of cytoplasmic messenger-like RNA not associated with polyribosomes as well as that of polyribosomal mRNA was investigated by labelling with [3H]uridine in conditions of arrested ribosomal RNA and mitochondrial RNA synthesis. The synthesis of ribosomal RNA was inhibited with toyokamycin and that of mitochondrial RNA with ethidium bromide. In both accumulation kinetics and actinomycin-D-chase experiments, cytoplasmic messenger-like ribonucleoprotein particles and polyribosomes were fractionated by buoyant density centrifugation in CsCl gradients. The half-life of free m1RNA was found to be of 1--2 h whereas the bulk of polyribosomal mRNA was stable over the time period considered (up to 8 h) but with a minor short-lived component. Purification of RNA from polyribosomes labelled under the same conditions and fractionation of it into polyadenylated and non-polyadenylated fractions showed that this short-lived minor component of half-life less than 1 h is non-polyadenylated.

Cytoplasm↗

PubMed1978

Frequency distribution of mRNA and pre-mRNA in growing and differentiated Friend cells.

The frequency distribution of poly(A)+-mRNA in growing and in differentiated Friend cells has been measured by mRNA-cDNA hybridization and their differences established by heterologous hybridization of mRNA of one type and cDNA of the other. It was shown that induction of Friend cells involves an increase in abundance of a small number of mRNAs, while no specific pattern of messenger disappearance could be detected. The frequency distribution of pre-mRNA was determined by hybridizing nuclear RNA with the cDNA probes complementary to mRNA. In uninduced Friend cells, it was shown that most precursor messenger sequences are present at a single frequency of about 3 molecules per nucleus, independently of their final frequency in polysomal mRNA. In induced Friend cells, the frequency distribution of pre-mRNA is more heterogeneous and correlated to some extent with the corresponding mRNA frequency distribution.

Animals↗

PubMed1976

Molecular-weight determination of animal-cell RNA by electrophoresis in formamide under fully denaturing conditions on exponential polyacrylamide gels.

A method for electrophoretic analysis of RNA under fully denaturing conditions on exponential gradient polyacrylamide gels is described. Full denaturation, and strand separation of DNA - RNA hybrids and double-stranded RNA is obtained in dry formamide only if electrophoresis is carried out at 45 degrees and 55 degrees C, respectively. In such conditions, the effects of secondary structure of RNA, important in aqueous medium, are suppressed and a linear correlation is obtained between the logarithm of the molecular weight of an RNA and its final position in the gel over the entire molecular weight range of 10(4) - 10(7). Based on absolute molecular weight standards, obtained from sequenced rRNA of Escherichia coli and tRNA and extrapolating to higher molecular weights the size of animal cell was reexamined. Precursor tRNA from HeLa cells migrates according to a molecular weight of 4.1 x 10(6). Nascent precursor mRNA has molecular weights of up to 5 x 10(6) in the case of duck erythroblasts and of up to 10(7) in HeLa cells. This seems to represent the largest size of non-viral animal-cell RNA molecules.

Animals↗

PubMed1976

Globin mRNA sequences in polyadenylated and nonpolyadenylated nuclear precursor-messenger RNA from avian erythroblasts.

Nuclear RNA from immature duck erythrocytes was fractionated into polyadenylated and nonpolyadenylated fractions, and globin mRNA sequences were determined by hybridization to DNA complementary to globin mRNA. 80--90% of labeled nuclear RNA is found to be nonpolyadenylated, and 70--80% of the globin mRNA sequences present in the nucleus are found in nonpolyadenylated molecules. These data suggest that polyadenylation does not specifically select for globin mRNA sequences. The nonpolyadenylated globin mRNA sequences present in the nucleus are found mostly in molecules of small size, close to the size of polyribosomal globin mRNA, suggesting that polyadenylation is a later event in globin mRNA formation.

Animals↗

PubMed1974

Synthesis and processing of nuclear precursor-messenger RNA in avian erythroblasts and HeLa cells.

The kinetics of synthesis and turnover of animal cell nuclear precursor-mRNA fractions all of which, in the case of avian erythroblast RNA, are shown by specific complementary DNA hybridization to contain globin mRNA sequences, were analyzed by exponential polyacrylamide gel electrophoresis. Three metabolically distinct size-fractions were characterized: (1) nascent precursor-mRNA (apparent molecular weight 5 to 20 x 10(6), approximate half-life 30 min), (2) intermediate-size precursor-mRNA (molecular weight 1 to 5 x 10(6), approximate half-life 3 hr), (3) small precursor-mRNA (molecular weight 0.5 to 1.5 x 10(6), half-life more than 15 hr). Nascent precursor-mRNA behaves kinetically as a precursor to the smaller precursor-mRNAs that accumulate in the nucleus, as well as to cytoplasmic mRNA; however, no stringent proof can be given that the two smaller nuclear precursor-mRNA fractions are direct physical precursors of functional mRNA. In terms of total mass, more precursor-mRNA accumulates in the nucleus than there is translated mRNA in the cytoplasm. Globin mRNA of final size (9 S) does not accumulate in the nuclei of avian erythroblasts.

Animals↗

PubMed1973