On the Synthesis of RNA in Lymphocytes Stimulated by Phytohemagglutinin

Abstract

The experimental conditions to study lymphocyte transformation in cultures derived from bovine lymph nodes are described. Both deoxyribonucleoprotein-bound and soluble RNA polymerase can conveniently be tested in cell lysates. During the course of transformation the activities of the two enzymes begin to rise a t the same time when the cells begin to accumulate stable RNA. At 30 h after addition of phytohemagglutinin both enzyme activities are enhanced by a factor of two over the controls.The transformation of small lymphocytes to large pyroninophilic cells under tho stimulus of phytohemagglutinin provides an excellent model system to study the conversion of resting cells to actively growing cells. The assumption that the synthesis of RNA may play a key role in this process has focused attention on the RNA metabolism in transforming lymphocytes [ 1 -81. A drastic increase in the incorporation of radioactive uridine into RNA soon after the addition of the stimulating agent phytoheniagglutinin indicated a rapid induction of RNA synthesis. The interpretation of these labelling experiments is difficult since the uptake of the labeled precursor into the intracellular nucleotide pool is increased considerably in lymphocytes treated with phytohemagglutinin [9,10]. Therefore it was decided to investigate the RNA synthesizing capacity of cell free systems derived from transforming lymphocytes. This approach avoids the discussion of "pool quesstions" and might give information as to what extent the RNA synthesizing apparatus of the cell is changed after stimulation.Different methods for studying RNA synthesis in cell free systems derived from animal cells have been described. Using bacterial RNA polymerase isolated chromatin has a template activity which changes with the physiological state of the cell [ll]. At low salt concentration isolated nuclei show RNA synthesizing activity which possibly reflects the activity of ribosomal operons within the nucleolus [12-151. The activity of cellular RNA polymerase bound to the chromatin can be tested at high salt concentration [16,17]. Finally, a soluble enzyme which uses exogeneous DNA as template for RNA synthesis can be extracted from the cells [is--211. The activities observed in these different assay systems probably reflect different aspects of tbe i n vivo conditions. Studies on the activity of chromatin-bound and of the soluble RNA polymerase will be described in this communication.I n order to perform such experiments large amounts of lymphocytes are needed. The limited supply of human blood used earlier [lo] prompted the investigation of other sources for lymphocytes. Bovine lymph node cells were found to respond to phytohemagglutinin in a similar way as human blood lymphocytes. These cells have been used throughout the studies. Culture conditions and the changes in leucine and uridine labelling as well as the net synthesis of DNA, RNA and protein after phytohemagglutinin stimulation will be described in the first section of this communication.Lymphocytes are particular...

Lectin‐Induced Changes among Polyadenylated and Non‐polyadenylated mRNA in Lymphocytes

“…Total cytoplasmic RNA was found to amount to 0.7 pg/ resting cell and to 0.9 pg/stimulated cell. These values are in agreement with published data [31]. We have used oligo(dT)-cellulose chromatography to separate polyadenylated RNA from non-polyadenylated RNA.…”

Polyamine accumulation during lymphocyte transformation and its relation to the synthesis, processing, and accumulation of ribonucleic acid

“…However, upon stimulation in vitro by Con A or phytohemagglutinin (Phaseolus vulgaris), lymphocytes are transformed into large blast cells capable of division (Robbins, 1964; Ling, 1968; Powell and Leon, 1970; Novogradsky and Katchalski, 1971). Among the early events in lymphocyte transformation are large increases in the rate of uptake of radioactive precursors and their incorporation into RNA and protein (Kay and Korner, 1966;Kay, 1968;Lucas, 1967;Hausen et al, 1969). This is followed by net RNA and protein accumulation at later times (Hausen et al, 1969;Fisher and Mueller, 1969;Forsdyke, 1967).…”

“…Among the early events in lymphocyte transformation are large increases in the rate of uptake of radioactive precursors and their incorporation into RNA and protein (Kay and Korner, 1966;Kay, 1968;Lucas, 1967;Hausen et al, 1969). This is followed by net RNA and protein accumulation at later times (Hausen et al, 1969;Fisher and Mueller, 1969;Forsdyke, 1967). DNA synthesis begins approximately 24 hr after stimulation (Powell and Leon, 1970; Novogradsky and Katchalski, 1971;Loeb and Agarwal, 1971) and the cells begin dividing on the second and third days of culture (Bender and Prescott, 1962).…”

Intercellular communication in phytohemagglutinin – induced lymphocyte agglutinates

“…Bovine lymphocytes were purified, cultivated, and stimulated with PHA-P according t o methods described elsewhere [4]. For the electrical measurements, t h e cells were sedimented and resuspended in a solution of Eagle's medium, containing 10 ' % calf serum, 0.6 % gelatine (Merck) and 0.8 % agar (Agar noble special, Difco).…”