G.David Novelli

Measurement of the incorporation of radioactive amino acids into protein by a filter-paper disk method

A rapid and sensitive method has been described for the measurement of amino acid incorporation in cell-free systems. Samples of a reaction mixture were acid-precipitated on filter-paper disks, washed and extracted to remove unincorporated radioactivity, and then counted directly in a liquid scintillation spectrometer. The method has been applied to both C14- and H3-labeled proteins.

Isoaccepting tRNA's in mouse plasma cell tumors that synthesize different myeloma protein

Abstract Different mouse plasma cell tumors have been shown to produce different homogenous species of myeloma immunoglobulin in large amounts and are currently thought to consist of monoclonal cells ( Potter et al. , 1965 ; Lennox and Cohn, 1967 ). We have previously observed multiple isoaccepting tRNAs in a tumor of this type, MOPC 31C ( Yang and Novelli, 1968a ). In the present work, comparative studies of isoaccepting tRNAs by co-chromatography on a reversed-phase chromatographic column have been performed on cell lines of transplantable mouse plasma cell tumors that produce different kinds of myeloma immunoglobulin. Similar chromatographic patterns were observed for most of the aminoacyl-tRNAs examined in these tumors. A marked difference, however, was found between the seryl-tRNAs of an immunoglobulin-A producer (MPC 62) and those of immunoglobulin-G producers (MPC 47 and MOPC 31C).

Isolation and properties of undermethylated phenylalanine transfer ribonucleic acids from a relaxed mutant of Escherichia coli

Abstract The function of methylated bases in transfer RNA (tRNA) was investigated with purified components of the phenylalanine-activating system. Reversed phase column chromatography was employed for the separation of tRNA Phe from mixtures of tRNAs synthesized by a relaxed mutant of Escherichia coli (met − ) before and after methionine starvation. Normal and undermethylated tRNA Phe were isolated and were characterized by their degree of methylation, amino acid-accepting capacity, and by end-group analysis. Kinetic data on initial velocities at saturating concentrations of tRNA indicated that the undermethylated species were considerably less reactive than the normal tRNA Phe .

Analysis of Isoaccepting tRNA's in Mammalian Tissues and Cells

Publisher Summary This chapter describes an application of the reversed-phase column chromatographic system (RPC); presents general procedures for preparing tRNA and aminoacyl-tRNA synthetases from the tissue and for preparing labeled aminoacyl-tRNA suitable for the chromatographic analysis; and demonstrates the analysis of iso-accepting tRNAs of an amino acid with a sample size in the range of 1 A260 unit of mixed tRNA. This method is very useful, especially in cases, where the source of material for preparing tRNA is limited, such as tissue culture cells and small animal organs. The sensitivity of this method is facilitated by the availability of radioactive amino acids with high specific activity. The isoaccepting peaks of an aminoacyl-tRNA are sufficiently well resolved in the column that quantitative comparative studies can be done by cochromatography of two samples with different labels. Because, the size of the tRNA sample is small, it becomes very important that artificial modifications of amino-tRNA should be prevented. The profiles of isoaccepting tRNAs for individual amino acids are much different between the mammalian and the bacterial sources.

Amino acid acceptor activity of enzymically altered soluble RNA from Escherichia coli

1. 1. Some amino acid acceptor functions of Escherichia coli s-RNA were shown to be quite resistant to Bacillus subtilis RNAase or RNAase T1 in the presence of Mg2+, whereas these functions were susceptible to bovine pancreatic RNAase under the same incubation conditions (RNAase, polyribonucleotide 2-oligonucleotidotransferase (cyclizing), EC 2.7.7.16). 2. 2. The resistance of individual amino acid s-RNAs for B. subtilis RNAase varied with different amino acids. The s-RNAs interacting with alanine, glycine, histidine, lysine, methionine, phenylalanine, tyrosine, valine and leucine were quite resistant to B. subtilis RNAase whereas the amino acid acceptor function of s-RNA specific for other amino acids such as arginine, aspartic acid, glutamic acid, proline, serine, threonine and tryptophan was rather sensitive to this RNAase. RNAase T1 gave results similar to those obtained with B. subtilis RNAase except that phenylalanine acceptor activity was inactivated. 3. 3. Specific activities for certain amino acyl RNAs were increased 2–3 times by treatment with B. subtilis RNAase. The base composition of a partially digested s-RNA differed from the original s-RNA in that it contained less guanine and adenine but more cytosine. The slope of the thermal denaturation curve, measured in the presence of Mg2+, was lower for resistant s-RNA than that of the undigested s-RNA. 4. 4. Partially digested s-RNAs were eluted from a column of methylated albumin of different salt concentration than untreated s-RNAs suggesting that relatively small alterations could be made in resistant s-RNAs without complete loss of acceptor activity for the two amino acids, phenylalanine and leucine.

In vitro amino acid incorporation into particle protein from maize seedlings

An amino acid incorporating system has been isolated from young maize seedlings. The addition of adenosine triphosphate, an adenosine triphosphate generator, guanosine triphosphate, MgCl2, KCl, and a soluble component are required for the incorporation of [I−14C]leucine into washed 105 000 × g centrifuged particles. The incorporation is rapid and almost exclusively into particle protein. Although the soluble component can be supplied by the pH 5 precipitate from rat liver, the maize 105 000 × g supernatant is required for full activity of the particles.

Restoration of aminoacylation activity of undermethylated transfer RNA by in vitro methylation

Abstract Undermethylated tRNA isolated from a RCrel mutant of Escherichia coli has been examined to determine whether its capacity to accept amino acids was comparable to that of normal tRNA from the same organism. Previously, little, if any differences have been reported (see Littauer and Milbauer, 1965 , Borek and Srinivasan, 1966 ). In this communication we report the restoration of aminoacylation activity of undermethylated tRNA by in vitro methylation. In a mixture of undermethylated tRNAs 2 from E. coli , we found that for four amino acids tested (Phe, Leu, Tyr, and His), the corresponding tRNAs showed decreased aminoacylation activities when compared to normal tRNA. Upon in vitro methylation of the undermethylated tRNAs with methylases free of RNase, an increase in the aminoacylation activities occurred. With two tRNA species (Phe and His), complete restoration of aminoacylation activity was observed.

A helical polysome model

Abstract Sedimentation rates at infinite dilution have been determined on eleven polysomes from rat liver. Frictional ratios derived from them have been used to estimate the axial ratios of the polysomes in solution. These indicate a configuration intermediate between a random chain and an ellipsoid of revolution. Three models were used to fit the data. The first, an ellipsoid of the same length and volume as the polysome, suggested that the structure being formed became more compact with increasing polysome size, assuming no change in hydration. The last two models showed that the rate of increase of axial ratio with polysome size was consistent with a 3- or 4-ribosome/turn helix of 18 or 20° pitch, respectively, assuming an increase in hydration of the polysome. The problems encountered in the polysome configuration were shown to be analogous to those encountered in fibrinogen, also a beadlike molecule.

Actinomycin D: Effect on the Immune Response

Actinomycin D injected simultaneously with sheep erythrocytes in female rats caused a delay in the immune response but had no effect on the rate or maximum amount of hemagglutinin produced. The delay was roughly proportional to the concentration of the antibiotic administered, and was up to 2 days for 75 �g in a 200-gram female rat (sublethal dose for females). The dose effect in the delay in response is consistent with the time when actinomycin would no longer be available to bind with newly synthesized DNA and when messenger-RNA production could occur. Similar results were obtained with another antigen, the enzyme β-galactosidase, in male rats during the secondary response.

Ribosome changes following illumination of dark-grown plants

Abstract The amino acid incorporating activity in vitro of ribosomes prepared from dark-grown plants is stimulated up to 200 % following exposure of the seedlings to low levels of light, and the percentage of polyribosomes is increased following this treatment. Red light is most effective in promoting this response. Polyuridylic acid (poly (U)) strongly enhances the incorporation of phenylalanine by ribosomes from both treated and control plants, and a portion of the observed stimulation by light is retained in the presence of an excess of this synthetic messenger.