Comparison of the in vitro translation capacity of Taenia crassiceps metacestode mRNA prepared by the phenol and cesium chloride method.
Total RNA of T. crassiceps metacestodes harvested from male and female NMRI mice was prepared by both the phenol extraction technique and cesium chloride (CsCl) gradient centrifugation. mRNA was selected by oligo (dT)-cellulose affinity chromatography and used as the template for the in vitro translation of parasite polypeptides in a cell-free rabbit reticulocyte lysate. The template activity of the mRNA obtained after CsCl preparation was clearly higher, as shown by the amount of 35S-methionine incorporated into the translation products and by fluorographed SDS-PAGE of the synthesized labelled polypeptides. SDS-PAGE fluorographs of antigens encoded by the mRNA prepared by CsCl centrifugation and selected by immunoprecipitation using purified IgG antibodies of T. crassiceps-infected mice (day 80 postinfection) exhibited seven labelled polypeptides of about 65, 46, 45, 42, 34, 29 kDa and a predominant 20-kDa antigen. The latter polypeptide was the only one recognized by the antibodies amongst the in vitro translation products directed by mRNA prepared by the phenol method.
Kleine-Herzbruch
R
R
Fachbereich Biologie der Philipps-Universität, Marburg/Lahn, Federal Republic of Germany.
Geyer
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E
D000818:Animals; D002499:Centrifugation, Density Gradient; D002586:Cesium; D002712:Chlorides; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008297:Male; D051379:Mice; D010636:Phenols; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification; D013621:Taenia / Q000235:genetics*
D002712:Chlorides; D010636:Phenols; D012333:RNA, Messenger; D002586:Cesium; C028019:cesium chloride