期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1979
卷号：76
期号：2
页码：625-629
DOI：10.1073/pnas.76.2.625
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：We report here a method of RNA preparation that may enrich for precursor RNA sequences and the results of an investigation of adeno-associated virus (AAV) RNA transcription that used this method. Whole cells were lysed with detergent and high salt and separated into supernatant and pellet (crude chromatin) fractions. These fractions were then separately deproteinized by proteolytic digestion and phenol extractions. DNA was removed from the preparation by two cycles of pancreatic DNase digestion and phenol extraction. Hybridization analyses of the RNA obtained from AAV/adenovirus-infected KB (human) cells revealed some AAV-specific RNA sequences that were not present in the mature 20S mRNA. These additional sequences were contained in AAV RNA molecules present in the pellet fraction, whereas the 20S AAV mRNA accumulated in the supernatant. A species of AAV-specific RNA (about 22S), which was associated only with the pellet fraction and was labeled only after a short pulse, appeared to have a kinetic relationship with the more stable cytoplasmic 20S mRNA. These putative AAV mRNA "precursors" and precursor sequences were not observed previously when conventional methods were used to obtain RNA from either whole cells or isolated nuclei.
