Biosynthesis of RNA in antibody-producing tissues.

These features, and in particular the problem of the control of antibody specificity, confer a special interest on the role of RNA biosynthesis and function in antibody-producing tissues. The present report concerns the biosynthesis of RNA in spleen and lymph nodes of rats immunized with H. pertussis, an antigen which produces in this species an unusually strong immune response. The isolation of a messenger RNA fraction and the study of its template activity have been reported elsewhere.1 Materials and Methods. -Sprague-Dawley rats of 200-300 gm were immunized by the injection of a mixture of killed H. pertussis (Eli Lilly Co.) and sheep red blood cells in the peritoneal cavity (i.p.) (4 X 109 H. pertussis, and 0.2 ml packed RBC) and in each footpad (1.5 X 109" H. pertussis, and 0.025 ml packed RBC); secondary immune response was initiated by injection of antigen at one fifth the initial dose. H3-uridine, H3-uracil, H3-D-L-leucine, C14-L-serine, and C14-L-valine were purchased from New England Nuclear Corp.; H3-thymidine was obtained from Schwarz Biochem, and carrierfree P32 from Oak Ridge National Laboratory. Polyvinylsulfate and 1,5-naphthalenedisulfonate were purchased from Eastman Organic Chemicals. Studies on in vivo synthesis of DNA, RNA, and proteins: Rats were killed either 8 hr following the i.p. injection of HU-thymidine (25 jc; 100 ic/jmole), or 2 hr following the i.p. injection of H3uracil (50 yc; 25 ic/imole) and H3-D-L-leucine (30 jc; 20 uc/imole). Spleen and lymph nodes (axillary and popliteal) were removed, weighed, and quickly frozen. Tissues were homogenized in 0.1 M Tris buffer pH 7 and precipitated in 0.25 N perchloric acid. The incorporation of radioactive precursors into DNA, RNA, and proteins was determined as described elsewhere.2 Extraction of RNA: Frozen tissues were homogenized at 4? C in a mixture of equal volumes of buffer (0.1 M Tris pH 5, 0.5% sodium dodecyl sulfate, and 0.5% naphthalenedisulfonate) and fresh phenol (Mallinckrodt, A. R., 88%, without preservative). The mixture was shaken for 6 rnin at 65? C and rapidly chilled. Following centrifugation and removal of the aqueous phase, phenol and interphase were re-extracted at 20? C with fresh buffer, and the pooled aqueous phases were treated twice more with half a volume of pheno:l at 20? C. RNA was precipitated once for 2 hr at 0? C in 0.1 M NaGC with 2.5 vol of chilled ethanol, and three times in 2 M K acetate with one third volume chilled ethanol for at least 2 hr at --20? C. Repeated precipitations under these conditions have been shown to free RNA from contamination by inorganic p32 (ref. 3), and oligodeoxyribonucleotides.4 The RNA pellet was dissolved in distilled water and stored at -20? C. When it was necessary to remove sRNA, additional precipitations in 2.5 M NaCl and in 0.2 M MgC25 were performed. For studies of labeled RNA, rats were killed at various times following intravenous injection of p32 (3-5 me) or H'-uridine (200 lc, 10 jc/jimole). Fractionation of RNA by sucrose gradient centrifugation: RNA preparations were centrifuged in a SW25 Spinco rotor at 25,000 rpm for 11 hr (except when otherwise indicated) at 15? C on sucrose gradients (5-20%) prepared in 0.005 M tris buffer (pH 7) containing 0.1 M NaCl and 0.5% 975