Use of Trypanosoma equiperdum infected rabbits as a source of splenic mRNA; construction of cDNA clones and identification of a rabbit mu heavy chain clone.
Rabbits were infected by Trypanosoma equiperdum and the splenic mRNA was isolated. In vitro translation of this RNA and immunoprecipitation with anti-light chain, anti-heavy chain, anti-mu and anti-VH antibodies demonstrated that T. equiperdum infection elicits large quantities of splenic mRNA encoding mu and kappa chains. The mu and gamma heavy chains and the kappa light chains synthesized in the cell-free translation system were specifically immunoprecipitated by antisera to heavy chain VHa and light chain kappa b allotypes. In vitro labeling of spleen cells from trypanosome-infected animals demonstrated that the biosynthetically labeled IgM has a mu chain of higher molecular weight than the mu chain synthesized by in vitro translation, a difference that is largely abolished when cellular glycosylation is blocked with the antibiotic tunicamycin. Enrichment for heavy chain or light chain mRNA was achieved by fractionating mRNA from trypanosome-infected animals on a sucrose gradient. cDNA clones carrying mu heavy chain sequences were produced using a 'one tube' protocol and identified by cross species hybridization and hybridization selection. Infection of rabbits with T. equiperdum followed by sucrose gradient enrichment of splenic mRNA has provided sufficient quantities of mRNA encoding mu heavy chain suitable for cDNA cloning.
D000818:Animals; D002474:Cell-Free System; D002999:Clone Cells / Q000378:metabolism; D004270:DNA, Circular / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000096:biosynthesis*; D011817:Rabbits; D013154:Spleen / Q000378:metabolism*; D014352:Trypanosomiasis / Q000276:immunology / Q000378:metabolism*
D004270:DNA, Circular; D007143:Immunoglobulin Heavy Chains; D012333:RNA, Messenger