Hybridization of poly(A)-containing ribonucleic acid transported in vitro to cyclic deoxyribonucleic acid-cellulose transcribed from cytoplasmic poly(A)-containing ribonucleic acid.
Isolated nuclei have been employed to study nucleocytoplasmic RNA transport in vitro; however, the specificity of the in vitro process remains largely undefined. To examine this specificity we have employed a hybridization technique that allows recovery of undegraded RNA after hybridization under stringent conditions. Cytoplasmic poly(A)-containing RNA was isolated from rat liver and used as a template for synthesis of cDNA-cellulose. Rat liver nuclei were isolated and incubated in vitro, and the released poly(A)-containing RNA was hybridized to the cDNA-cellulose transcribed from authentic cytoplasmic RNA. A significant portion hybridized (18.5 +/- 4.4%), comparable to the portion of homologous cytoplasmic poly(A)-containing RNA that hybridized (20.4 +/- 2.3%), indicating a considerable sequence homology between these populations. Similar results were obtained when cDNA was transcribed from RNA which was transported in vitro in 45S ribonucleoprotein (21.6%). Control nuclear RNA hybridized to a significantly smaller extent (9.1 +/- 2.7%), and the percentage hybridizing decreased markedly under conditions that allowed nuclear RNA processing but not transport (to 4.3 +/- 2.1%), showing that the hybridization was not due to nonspecific leakage of nuclear RNA. This technique should prove valuable for following in vitro processing of specific probes and for enrichment of sequences differentially transported in vitro following acute injury or carcinogen treatment of rats.
D000818:Animals; D001692:Biological Transport; D002482:Cellulose / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004270:DNA, Circular; D008099:Liver / Q000032:analysis; D008297:Male; D009693:Nucleic Acid Hybridization; D011061:Poly A / Q000235:genetics*; D012313:RNA / Q000235:genetics* / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains; D014158:Transcription, Genetic
D004270:DNA, Circular; D011061:Poly A; D012313:RNA; D002482:Cellulose