Mary G. Hamilton

Nucleoplasmic ribonucleoprotein particles of rat liver: II. Physical properties and action of dissociating agents

Abstract The physical properties of rat liver nuclear particles that contain DNA-like RNA have been studied by various ultracentrifugal techniques. The nuclear particles were resolved into three fractions (b, c and d) by zone centrifugation. When submitted to sedimentation velocity analysis, these particles showed considerable polydispersity and had weight-average sedimentation coefficients of 43 S (for b particles), 81 S (for c particles) and 100 S (for d particles). Analysis by high speed equilibrium centrifugation gave molecular weights of 3.3 · 10 6 , 6.0 · 10 6 , and 11.0 · 10 6 daltons for b, c and d particles. This suggests that the nuclear particles must have highly elongated or irregular shapes. Their stability to various agents has been investigated. While neither high salt concentrations nor Triton X-100 had any effect, sodium deoxycholate disrupted the structure. The proteins of the particles have been examined by two electrophoretic techniques. In sodium dodecyl sulfate gels the protein moiety showed many bands of different molecular sizes although the pattern obtained in acetic acid-urea gels was relatively simple. Treatment with sodium deoxycholate resulted in changes in the band patterns.

The purification and properties of ribosomes from the thymus nucleus.

Abstract Methods are described for the isolation and purification of ribonucleoprotein particles (ribosomes) from isolated calf-thymus nuclei. About half of the total ribonucleic acid of the nucleus can be recovered in the purified ribosome fraction. Nuclear ribosomes can be purified by centrifugation in a sucrose density-gradient. The ribosome peak contains about 36% RNA. Treatment of the peak material with deoxycholate removes much of the protein. The resulting ribosomes have a sedimentation coefficient of 78 S and about 60% of their mass is RNA. The average base composition of this ribosomal RNA is given and compared with that of other nuclear RNA fractions. The changes in state of the nuclear 78-S ribosome with changing pH, ionic strength, and Mg 2+ concentration are described. Electron microscopy of isolated ribosomes after shadow-casting with chromium indicates that the 78-S ribosomes are about 200 A in width.

Nucleoplasmic ribonucleoprotein particles of rat liver: I. Selective degradation by nuclear nucleases

Abstract Nucleoplasm isolated from non-growing rat livers contains a complex mixture of ribonucleoprotein particles, none of them mature ribosomes, polysomes or poly-somal-like structures. Approx. 35% of the rapidly labeled RNA was found in these particles, while the rest remained bound to the chromatin. These particles contain approx. 80% protein, 20% RNA and less than 1% DNA. The buoyant densities range between 1.38 and 1.41 g/cm 3 in CsCl. In a sucrose gradient fractionation three broad peaks are obtained with average S values of 60, 80 and 110. The two more rapidly sedimenting peaks are selectively digested by nuclear nucleases and pancreatic ribonuclease. RNA-DNA hybridization experiments with competition suggest that the 60-S peak is transferred to the cytoplasm.

The use of guanidine-HCl for the isolation of both RNA and protein from RNA tumour viruses.

The RNA components of two C-type RNA viruses, avian myeloblastosis virus and Friend leukaemia virus, have been isolated by treatment of the viruses with 6 M-guanidine-HCl and precipitation with ethanol. The virus proteins were recovered by lyophilization of the guanidine-HCl-ethanol supernatant after thorough dialysis against 0.5 mM-dithiothreitol. This simple method yielded RNA of similar quality to the phenol and sodium dodecyl sulphate (SDS) extraction methods, and the same amount of 60-70S RNA, although a fraction of the smaller (4S) species remained in the protein fraction. The sedimentation patterns of heat-denatured RNA extracted by either method were similar. Electrophoretic analyses of the extracted proteins in polyacrylamide gel gradients containing SDS gave patterns that were very similar to those obtained by direct analysis of SDS disrupted viruses.

The basic proteins of Aspergillus fumigatus with tumor-inhibiting properties.

Abstract 1. 1. The factor in culture filtrates of Aspergillus fumigatus responsible for tumor inhibition in mice has been partially purified by ethanol fractionation and electrophoretic separation. 2. 2. Both the activity and the toxicity were associated with a highly basic protein or group of proteins with isoelectric points near pH 10.0.

5.3 S RNA is a discrete cleavage product from the 5'-terminus of 18 S RNA of rat liver ribosomes.

Abstract A 5.3 S RNA species observed in urea-gel electrophoretic analysis of the RNA of the small ribosomal subunit of rat liver has been identified from its sequence as the 5′-terminal 133–134 base fragment of 18 S RNA. Presumably it is cleaved by an endogenous endonuclease when the ribosomal subunits are dissociated, because it usually is not observed in 18 S RNA obtained by direct extraction of cells or tissues.