We describe a simple procedure for isolating specific mRNAs and for mapping them to the regions of the DNA from which they originate. The method involves hybridization of total cytoplasmic RNA to restriction fragments of DNA that have been fractionated in agarose gels and immobilized on nitrocellulose filters. The hybridization-selected RNAs are eluted and translated in a cell-free system in order to identify their encoded polypeptides. Optimal hybridization and filter washing conditions are given for selection of mRNAs from adenovirus 2-infected cells and transformed cells.Synthesis of individual polypeptides directed by purified mRNAs in a cell-free translation system provides compelling evidence for the expression of specific genes. The ability to purify functional mRNAs is a prerequisite for understanding some of the detailed mechanisms involved in gene regulation. Characterization of purified mRNAs by cell-free translation, in combination with other techniques, can yield information about the spatial and topological arrangement of transcripts along the genome, the location of protein coding regions within the mRNA, mRNA structure, and mRNA abundance.Several methods for isolating specific mRNAs have been developed. DNA, either in liquid or bound to immobilized supports, has been used to hybridize and thereby sequester specific mRNAs from the plethora of cellular RNA species. These methods have included isolation of RNA-DNA hybrids by: selective binding to hydroxylapatite (1); selective exclusion through agarose (2) or Sepharose 4B (3); the use of DNA covalently bound to cellulose (4, 5) or Sepharose (6); DNA bound directly to nitrocellulose (7-9); DNA enzymatically synthesized and covalently bound to oligo(dT)-cellulose (10).Here we describe an efficient method by which specific mRNAs can be purified and used to determine the location of these mRNAs with respect to their DNA coding regions. This method relies on hybridization of total cytoplasmic RNA to restriction fragments of DNA which have been immobilized on nitrocellulose filters. The hybridization-selected mRNAs are eluted from the DNA and identified by the polypeptide products that are synthesized in a reticulocyte cell-free system. The map positions of the mRNA transcripts on the DNA can be determined directly because the genomic coordinates of the DNA restriction fragments are known.This procedure has several advantages. Most importantly, purification of the DNA restriction fragments is not required. Rather, DNA restriction fragments are fractionated by electrophoresis in agarose gels and then directly transferred to the nitrocellulose filter membrane by the method of Southern (11). In this way, many different DNA fragments may be easily handled in a single experiment. In addition, the nitrocellulose filters that contain the bound DNA fragments can be used several times. By this procedure, it is possible to identify the translation product of a specific mRNA which is otherwise obscured by the large number of polypeptides that are synthesized...

Simian adenovirus 7 (SA7) is a highly oncogenic virus, capable of causing tumors in hamsters upon the direct injection of viral DNA. We determnined the transcriptional organization of the transforming region and compared it with that of the human adenoviruses. This analysis demonstrated that there are two independently promoted transcription units similar to the Ela and Elb regions of the human adenoviruses. The nucleotide sequence of the SA7 Ela region demonstrated considerable homology with the human adenoviruses, both in the sequences that regulate Ela expression and in the encoded polypeptides. The amino acid homology was reflected in the ability of SA7 to complement the growth of human adenoviruses mutant in the Ela region. Furthermore, we found two regions of amino acid homology unique to SA7 and the highly oncogenic human adenovirus 12.

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Adenovirus early region 1A (ETA), which gives rise to three overlapping transcripts, was inserted into a murine leukemia virus-derived vector, and recombinant viruses were used to prepare permanent cell lines of NIH 3T3 cells containing DNA copies of the individual 13S, 12S, and 9S mRNAs. Integrated proviral copies of the recombinant genomes were rescued as bacterial plasmids from each of the cell lines, and the DNA sequence of ETA was demonstrated to be a precise copy of the individual transcripts. The DNA copies were shown to be expressed as part of the full-length retroviral transcript by ST nuclease analysis, and the synthesis of their encoded polypeptides was demonstrated by immunoprecipitation. Those cell lines expressing the polypeptide encoded by the 13S transcript were shown to contain that function required for regulating the accumulation of mRNAs from adenovirus early genes by their ability to complement the adenovirus type 5 ElA deletion mutant d1312. Cell lines expressing polypeptides encoded by the 13S, 12S, and 9S transcripts showed characteristic alterations in morphology. Two-dimensional gel electrophoresis of total cellular protein derived from the three cell lines demonstrated that each ElA gene product elicits specific alterations in the patterns of proteins expressed. Studies of the expression of two specific genes, those encoding fibronectin and collagen type 1, indicated that the observed alteration in levels of the two proteins results from a reduction in RNA levels induced by ETA functions.