Improved preparation of poly(A) Sepharose and the isolation of oligo(U) containing heterogeneous nuclear RNA from Friend erythroleukemic cells.
When poly(A) sepharose (prepared according to previously published procedures) was stored in aqueous buffer at 4 degrees C for 5 days or longer, it bound nonspecifically a high percentage of the input RNA which could not be eluted with formamide. We have found that treatment with ethanolamine, followed by dehydration with ethanol yielded poly(A) sepharose which was stable for many months and possessed a low degree of nonspecific binding. Chromatography on poly(A) sepharose permitted the specific isolation of that fraction of Friend erythroleukemic cell heterogeneous nuclear RNA (hnRNA) which contained oligo(U) sequences. Approximately 10% of the hnRNA which contained a poly(A) sequence [poly(A+)] also contained an oligo(U) sequence. Interestingly, prior HCHO denaturation of the hnRNA enhanced binding of the poly(A+) oligo(U+) hnRNA to poly(A) sepharose by tenfold. This suggested that the oligo(U) sequence may be in a region with secondary structure, possibly an intramolecular duplex with the 3' poly(A). Friend cell oligo(U) sequences ranged from 20 to 50 nucleotides in length and, thus, were similar to the oligo(U) sequences which heretofore had only been shown to be present in HeLa cell hnRNA. These results established that rodent cell hnRNA contain oligo(U) sequences and demonstrate, for the first time, that hnRNA containing both a poly(A) and an oligo(U) sequence can be separated from other classes of hnRNA. In addition, conditions are presented for the removal of HCHO from nucleic acid.
D000818:Animals; D002847:Chromatography, Agarose; D004915:Leukemia, Erythroblastic, Acute; D007942:Leukemia, Experimental / Q000032:analysis*; D009841:Oligonucleotides / Q000302:isolation & purification*; D009843:Oligoribonucleotides / Q000302:isolation & purification*; D011134:Polysaccharides; D012332:RNA, Heterogeneous Nuclear / Q000302:isolation & purification*; D012334:RNA, Neoplasm / Q000302:isolation & purification*; D012685:Sepharose / Q000031:analogs & derivatives; D014500:Uracil Nucleotides / Q000302:isolation & purification*
D009841:Oligonucleotides; D009843:Oligoribonucleotides; D011134:Polysaccharides; D012332:RNA, Heterogeneous Nuclear; D012334:RNA, Neoplasm; D014500:Uracil Nucleotides; C027916:oligo(U); C030810:poly A-agarose; D012685:Sepharose