Immunochemical isolation of gamma-globulin mRNA and estimation of immunoglobulin gene reiteration.
The polyribosomes synthesizing gamma-globulin have been isolated by the achievement of specific precipitation using bentonite-treated anti-IgG antibody. The RNA extracted from the immunochemically precipitated polysomes was tested for its ability to direct the synthesis of proteins in a cell-free system. The specific gamma-globulin-synthesizing activity (cpm of gamma-globulin synthesized/microgram RNA) of this RNA was 10-fold greater than that from total polysomes. gamma-globulin mRNA (messenger RNA) isolated by immunoprecipitation was more than 89% pure with respect to contamination by other species of mRNA. The products synthesized by the cell-free system were also analyzed by sodium dodecyl sulphate(SDS)-polyacrylamide gel electrophoresis. This RNA has been hybridized with mouse myeloma DNA. The estimation of immunoglobulin gene reiteration was carried out using hybridization kinetics with consideration given to the DNA/RNA ratio since the estimation from the "half Cot value" is not accurate. The results suggest that in the mouse there are about 20 copies per subgroup of genes coding for the variable region of the H and L chains.
D000483:Alleles; D002460:Cell Line; D002474:Cell-Free System; D005796:Genes; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics*; D009194:Myeloma Proteins / Q000096:biosynthesis / Q000235:genetics; D011132:Polyribosomes / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification*
D007074:Immunoglobulin G; D009194:Myeloma Proteins; D012333:RNA, Messenger