Key Experiment: The Discovery of Introns

Spliced Segments at the 5′ Terminus of Adenovirus 2 Late mRNA

Susan M. Berget, Claire Moore, and Phillip A. Sharp

Massachusetts Institute of Technology, Cambridge, Massachusetts

Proceedings of the National Academy of Sciences USA, Volume 74, 1977, pages 3171-3175

The Context

Prior to molecular cloning, little was known about mRNA synthesis in eukaryotic cells. However, it was clear that this process is more complex in eukaryotes than in bacteria. The synthesis of eukaryotic mRNAs appeared to require not only transcription, but also processing reactions that modify the structure of primary transcripts. Most notably, eukaryotic mRNAs appeared to be synthesized as long primary transcripts, found in the nucleus, which were then cleaved to yield much shorter mRNA molecules that were exported to the cytoplasm.

These processing steps were generally assumed to involve the removal of sequences from the 5′and 3′ends of the primary transcripts. In this model, mRNAs embedded within long primary transcripts would be encoded by uninterrupted DNA sequences. This view of eukaryotic mRNA was changed radically by the discovery of splicing, made independently by Berget, Moore, and Sharp, and by Louise Chow, Richard Gelinas, Tom Broker, and Richard Roberts (An amazing sequence arrangement at the 5′ends of adenovirus 2 messenger RNA, Cell 12: 1-8, 1977).

The Experiments

Both of the research groups that discovered splicing used adenovirus 2 to investigate mRNA synthesis in human cells. The major advantage of the virus is that it provides a model that is much simpler than the host cell. Viral DNA can be isolated directly from virus particles, and mRNAs encoding the viral structural proteins are present in such high amounts that they can be purified directly from infected cells. Berget, Moore, and Sharp focused their experiments on an abundant mRNA that encodes a viral structural polypeptide known as the hexon.

To map the hexon mRNA on the viral genome, purified mRNA was hybridized to adenovirus DNA and the hybrid molecules were examined by electron microscopy. As expected, the body of the hexon mRNA formed hybrids with restriction fragments of adenovirus DNA that had previously been shown to contain the hexon gene. Surprisingly, however, sequences at the 5′end of hexon mRNA failed to hybridize to DNA sequences adjacent to those encoding the body of the message, suggesting that the 5′end of the mRNA had arisen from sequences located elsewhere in the viral genome.

This possibility was tested by hybridization of hexon mRNA to a restriction fragment extending upstream of the hexon gene. The mRNA-DNA hybrids formed in this experiment displayed a complex loop structure (see figure). The body of the mRNA formed a long hybrid region with the previously identified hexon DNA sequences. Strikingly, the 5′ end of the hexon mRNA hybridized to three short upstream regions of DNA, which were separated from each other and from the body of the message by large single-stranded DNA loops. The sequences at the 5′end of hexon mRNA thus appeared to be transcribed from three separate regions of the viral genome, which were spliced to the body of the mRNA during the processing of a long primary transcript.

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