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Biomedical subjects

T Pederson

Publications and source records attributed to T Pederson.

At least 109 records · Page 6Linked to original sources
PubMed1976

Poly (A)-rich ribonucleoprotein complexes from HeLa cell messenger RNA.

Polyribosomal messenger RNA from HeLa cells contain 3'-OH-terminal polyadenylate sequences approximately 133 nucleotides in length (weight average). When analyzed at the ribonucleoprotein level of organization these poly(A)-rich sequences are found to contain tightly bound proteins. These proteins remain associated with the poly(A)-rich RNA during affinity chromatography of RNase A and T1-digested polyribosomes on poly(U)-Sepharose in 0.5 M NaCl, and co-elute from the column with the RNA at 50% formamide. Controls establish that the co-purification of the proteins with poly(A) on poly(U)-Sepharose requires the molecular integrity of the poly(A). Polyacrylamide gel electrophoresis resolves the poly(A)-specific proteins into two components of 74,000 and 62,000 molecular weight. The larger protein is the same size as that previously reported to be associated with poly(A)-rich sequences in HeLa heterogeneous nuclear RNA (Kish, V.M., and Pederson, T. (1975), J. Mol. Biol. 95, 227-238). It is concluded that both HeLa nuclear and polyribosomal poly(A) sequences have a protein (62,000 molecular weight) associated with poly(A) appears to be confined only to messenger RNA.

Chromatography, Affinity↗
PubMed1976

Rapid, preparative-scale purification of chromatin proteins.

Methods are desceibed which permit rapid isolation of chromatographically purified histone and non-histone chromatin proteins under relatively mild chemical conditions. Chromatin is isolated from purified nuclei, dissociated in guanidine - HCl-urea and the nucleic acids removed by ultracentrigugation. This can be accomplished in 10 h by employing maximum-force rotors (500 000 x g). The proteins are then fractionated by a batch ion-exchange method, which leads to a rapid and complete separation of the histones and non-histone components, in apparently undegraded form. With these methods it is possible to obtain mg quantities of chromatographically pure histone and non-histone proteins in less than a single working day.

Binding Sites↗
PubMed1975

A special class of non-histone protein tightly complexed with template-inactive DNA in chromatin.

A special class of non-histone protein ("tight protein") is identified in purified HeLa cell chromatin on the basis of its failure to dissociate from the DNA at very high ionic strength (2.5 M NaCl-5.0 M urea), where over 92% of the total chromatin protein is released. The tight proteins are insoluble in 0.4 N H2SO4 and lack histones as determined by polyacrylamide gel electrophoresis. They have molecular weights between 14,000 and 85,000 with over 70% of the polypeptide chains between 14,000 and 30,000 mol wt. This is the same size range as the non-histone proteins which others have found to display species-specific DNA binding in vitro. There is approximately one molecule of tight protein per 275 DNA base pairs. The tight proteins are characterized by much higher rates of labeling with amino acids than the histones and non-histone chromatin proteins that are dissociated from the DNA by high ionic strength, but they have the lowest phosphorylation levels. Chromatin fractionation experiments were performed to investigate the distribution of tight proteins between template-active and template-inactive regions. Under specific conditions, spleen DNase (DNase II) selectively shears those portions of HeLa cell chromatin that contain nascent RNA transcripts. This nascent RNA-enriched chromatin fraction also contains a high level of the proteins known to be complexed with heterogeneous nuclear RNA in ribonucleoprotein particles and contains over 70% of the RNA polymerase activity of total chromatin. When this method was employed to investigate the distribution of tight proteins, they were found to be almost entirely confined to the template-inactive fraction. Although these experiments do not elucidate the precise function of these proteins, they identify, for the first time, a particular subclass of non-histone chromosomal protein which is distributed asymmetrically between transcriptionally active and inactive chromatin regions.

Binding Sites↗
PubMed1975

Metabolic stability of messenger ribonucleoprotein in HeLa cells.

The proteins bound to HeLa cell polyribosomal messenger RNA were isolated by subjecting salt-washed, puromycin-disassembled polyribosomes to a limited digestion with pancreatic ribonuclease (ref. 1, Auerbach, S. and Pederson, T. (1975) Biochem. Biophys. Res. Commun. 63, 149-153). Label-chase experiments with radioactive amino acids revealed that the in vivo decay kinetics of the messenger RNA-associated proteins were approximately first-order, with t1/2 equal 13-15 h. The results suggest that HeLa messenger RNA and its specific set of associated proteins do not behave as single units metabolically.

Drug Stability↗
PubMed1975

Ternary solvents to investigate proteins at zub-zero temperatures.

Mixtures of water, ethylene glycol and methanol in different volume ratios have been selected to carry out kinetics of enzyme reactions at sub-zero temperatures with the intention to reduce maximally the viscosity. Density, viscosity and dielectric constant values of these mixtures as a function of temperature are reported, as well as values of the protonic activity of several buffers under such conditions. A procedure to avoid or delay the eventual damaging effect of methanol on proteins is described.

Acetates↗
PubMed1975

Ribonucleoprotein particles containing heterogeneous nuclear RNA in the cellular slime mold Dictyostelium discoideum.

As in higher eukaryotes, heterogeneous nuclear RNA (HNRNA) in the cellular slime mold Dictyostelium discoideum is associated with proteins in the form of ribonucleoprotein particles. Mixing experiments with deproteinized hnRNA establish that the nuclear ribonucleoprotein particles are not formed artificially during isolation. In contrast to comparable material from mammalian cells (polydisperse, 40-25- S), Dictyostelium heterogeneous nuclear ribonucleoprotein particles sediment at only 55 S in sucrose gradients, possibly reflecting the smaller size of slime mold hnRNA relative to the large hnRNA found in higher eukaryotes. The RNA of the nuclear 55S ribonucleoprotein particles is shown to be hnRNA by virtue of its size (15S), content of polyadenylate sequences, and hybridization kinetics at DNA excess. The hnRNA-associated porteins are electrophoretically complex and have molecular weights between 20,000 and 150,000. In 0.35 M NaCl most of the proteins are released from the hnRNA. However, a single protein of 72,000-74,000 molecular weight remains bound, as indicated by its co-chromatography with the RNA on poly(U)-Sepharose and banding in Cs2SO4. The same protein is recovered when heterogeneous nuclear ribonucleoprotein is digested with T1 ribonuclease under conditions where the poly(A) is nuclease-resistant. The 73,000 molecular weight protein appears to be specifically bound to polyadenylate sequences in Dictyostelium hrRNA.

Adenine↗
PubMed1974

Gene activation in eukaryotes: are nuclear acidic proteins the cause or the effect?

Nuclear acidic proteins have been implicated in the positive control of gene transcription in eukaryotes. This hypothesis was examined in greater detail by analysis of these proteins during experimental gene activation by a technique for fractionating nuclei into chromatin and the ribonucleoprotein particles that contain heterogeneous nuclear RNA. When synthesis of rat-liver heterogeneous nuclear RNA was stimulated by administration of hydrocortisone, there was a parallel increase in the labeling of acidic proteins in ribonucleoprotein particles. However, there was no detectable effect on the labeling of either acidic chromatin proteins or histones. Thus, the nuclear acidic proteins that respond to the hormone are concerned with a post-transcriptional event, namely the assembly and processing of ribonucleoprotein particles that contain heterogeneous RNA, rather than with direct gene activation. Increases in synthesis of "chromatin" acidic proteins during gene activation observed by others may reflect the presence of these ribonucleoprotein particles in crude chromatin preparations.

Animals↗
PubMed1972

Nonhistone chromosomal proteins in synchronized HeLa cells.

Chromatin was isolated from synchronized HeLa cells at different stages of the cell division cycle and fractionated into DNA, histones, and nonhistone proteins. Electrophoresis of the nonhistone proteins in sodium dodecyl sulfate-polyacrylamide gels revealed a highly reproducible pattern of 22 bands, having estimated molecular weights of 15,000-180,000, with 85% (by mass) over 40,000. The amounts of some nonhistone proteins varied during the cell cycle by as much as 50%, while others remained at a constant level. One group of nonhistone proteins (molecular weight 75,000) was greatly reduced just before the start of DNA replication (S-phase), then returned to normal levels in the mid-S phase. These results are discussed with regard to the possible role of nonhistone proteins in regulating chromosome structure and function.

Carbon Isotopes↗
PubMed1972

Chromatin structure and the cell cycle.

Pancreatic DNase I is used to probe the structure of chromatin isolated from synchronized HeLa cells. The degree to which DNA in chromatin is protected from DNase attack varies during the G(1), S, and G(2) phases of the cell cycle. In addition, the DNase sensitivity of chromatin from contact-inhibited African green monkey kidney cells differs from that of actively dividing, subconfluent cultures. These cell cycle-dependent chromatin changes were observed consistently at all enzyme concentrations (5000-fold range) and incubation times (15 min-2 hr) tested. The results indicate that the degree of complexing between DNA and chromosomal proteins changes during interphase, and they suggest that the chromosome coiling cycle of visible mitosis may extend in more subtle form over the entire cell cycle.

Animals↗
PubMed1972

Chromatin structure and the cell division cycle. Actinomycin binding in synchronized HeLa cells.

Measurements of actinomycin-(3)H binding in synchronized HeLa cells reveal that the binding capacity of chromatin decreases progressively during the S phase despite a doubling of nuclear DNA content, reaches a minimal level during G(2) and mitosis, and then increases gradually throughout the subsequent G(1) interval. Since this pattern was evident in experiments with living cells, ethanol-fixed cells, and isolated nuclei, but not with purified DNA, the actinomycin binding profile may reflect changes in the degree of association between DNA and chromosomal proteins at different stages of the cell cycle.

Carbon Isotopes↗