Sequence content of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells.
Oligo(uridylic acid)-containing [oligo(U+)] RNA was isolated from poly(adenylic acid)-containing [poly(A+)] mRNA from HeLa cells by using either formaldehyde pretreatment or poly(A) removal, both of which resulted in increased accessibility of oligo(U)-rich sequences to a poly(A)-agarose affinity column. In this report, we compared the sequence content of oligo(U+) RNA with that of molecules lacking oligo(U) [oligo(U-) RNA] by their relative hybridization to cDNA reverse-transcribed from poly(A+) mRNA and by comparison of their in vitro translation products synthesized in a rabbit reticulocyte lysate. Formaldehyde-modified poly(A+) RNA, treated to remove the formol adjuncts, was inactive as a template for in vitro protein synthesis; consequently, only depolyadenylated RNA, which retains its translatability, could be used in the translation studies. The hybridization kinetic experiments revealed that oligo(U+) RNA contained most of the sequence information present in oligo(U-) RNA but at a reduced level (ca. 25%), the majority of the oligo(U+) RNA sequences being poorly represented in the cDNA. This result was supported by one- and two-dimensional gel analysis of their in vitro translation products which showed that oligo(U+) RNA, although less effective as a template for translation than oligo(U-) RNA, coded for proteins, the most abundant of which were encoded by rare messages not highly represented in oligo(U-) RNA or the total poly(A+) RNA. Although some minor products were synthesized by both oligo(U+) and oligo(U-) RNA, at least 33 proteins were unique to or highly enriched in the pattern of products directed by oligo(U+) RNA.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005557:Formaldehyde; D006367:HeLa Cells / Q000032:analysis; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D014500:Uracil Nucleotides / Q000235:genetics / Q000302:isolation & purification*
D012333:RNA, Messenger; D014500:Uracil Nucleotides; C032369:oligo(U+)RNA; D005557:Formaldehyde