Isolation and characterization of ovine prolactin and growth-hormone messenger RNAs.
Methods are described in this paper for obtaining and characterizing highly enriched preparations of ovine prolactin (PRL) and growth-hormone (GH) messenger RNAs. Purification steps include phenol extraction, oligo-(dT)-cellulose chromatography, sucrose-gradient ultracentrifugation, and polyacrylamide-gel electrophoresis. This sequence of procedures results in messenger preparations that are about 92% pure for prolactin mRNA and 78% pure for the growth-hormone mRNA species. Thus, these two closely related mRNAs can be isolated from the same tissue source at a purity adequate for cloning and nucleic acid hybridization experiments. Translation experiments with the cap analogue 7-methylguanosine, and end-labeling of the nucleic acid before and after beta-oxidation indicate that both messages possess blocked 5'-termini, and that these are part of previously described cap structures. Polyadenosine tracts of 30-160 residues were found at the 3'-ends of both purified species. Finally, sizing experiments suggest both mRNAs contain approx. 30% more bases than accounted for by the coding regions and the poly(A)-tracts. Their physical characteristics thus agree with those of most eucaryotic messages to date.
D000818:Animals; D001482:Base Composition; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D013006:Growth Hormone / Q000302:isolation & purification*; D007158:Immunologic Techniques; D010903:Pituitary Gland, Anterior / Q000032:analysis; D011388:Prolactin / Q000302:isolation & purification*; D014176:Protein Biosynthesis; D012314:RNA Cap Analogs / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism; D012756:Sheep; D013053:Spectrophotometry
D012314:RNA Cap Analogs; D012333:RNA, Messenger; D011388:Prolactin; D013006:Growth Hormone