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An Amazing Sequence Arrangement at the 5’ Ends of Adenovirus 2 Messenger
RNA
Louise T. Chow, Richard E. Gelinas, Thomas R. Broker and Richard J. Roberts
Spliced Segments at the 5’ Terminus of Adenovirus 2 Late
mRNA
Susan M. Berget, Clair Moore, and Phillip A. Sharp
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Background:
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A Little History. . .
• In 1977, research groups working on the expression of adenovirus late genes at MIT and at Cold Spring Harbor reported that the viral mRNAs were:
• "mosaic molecules consisting of sequences complementary to several non-contiguous segments of the viral genome".Quote from Adenovirus amazes at Cold Spring Harbor (1977) Nature 268: 101-104.
• Phil Sharp (at MIT) and Rich Roberts (at Cold Spring Harbor) led the research groups which made this discovery. They shared the Nobel Prize in Medicine (1993) for their discovery.
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This “Mosaic” Property Was Soon Found to be a General Property of Euakaryotic mRNAs
The notion of the cistron, the genetic unit of function that one thought corresponded to a polypeptide chain, now must be replaced by that of a transcription unit containing regions which will be lost from the mature messenger -- which I suggest we call introns (for intragenic regions) -- alternating with regions which will be expressed -- exons. The gene is a mosaic: expressed sequences held in a matrix of silent DNA, an intronic matrix. Gilbert, W. (1978) Why genes in pieces? Nature 271: 501
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RNA Modification and Processing Review• The initial nuclear RNA copy of protein-coding
genes transcribed in eukaryotes is known as heterogeneous nuclear RNA (but it’s too large!)
• hnRNA is processed while it is being synthesized and before it leaves the nucleus en route to the ribosomes located in the cytoplasm. Three types of modification are made:
• The poly(A) tail is added to the 3' end.• A cap is added to the 5' end.• Introns are spliced out.
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An more extreme example is that of the dystrophin gene. The initial pre-mRNA transcript could be > 2 million nt in size but this is spliced down to an mRNA of 14,000 nt by the removal of 78 introns
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The Discovery of Introns was Made Through Electron Microscopy using R-Loop Analysis
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Spliced Segments at the 5’ Terminus of Adenovirus 2 Late mRNA
Susan M. Berget, Clair Moore, and Phillip A. Sharp
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The Researchers are Trying to Understand the Eukaryotic Transcription Process• The researchers in both studies elected to use a
simple system to study this: Adenovirus 2
• Adenovirus 2 has features comparable to its eukaryotic host (human cells) namely the presence of long polyadenylated transcripts in the host nucleus, but only a small percentage of this nuclear RNA appears as polyadenylated mRNA on cytoplasmic polysomes – similar to heterogeneous nuclear RNA
• Polysome =The assemblage of mRNA, ribosomes, and the growing peptide chain present during translation.
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Experiments
• Isolated a Late Stage Ad2 mRNA (Hexon)
• Hybridized to Restriction Fragments of Ad2 DNA (Eco R1, and Hind III)
• Used R-Loop technique coupled with Electron Microscopy to Identify non-hybridizing segments and map position
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Observations & Conclusions• A non-hybridizing 5’ RNA tail (160 nts) was nearly
always present in hexon – Hind III fragment A (which should entirely contain the hexon mRNA) R-Loops (Here hexon mRNA hybridized with ds DNA fragment)
• This unexpected anomaly is still present when using ssDNA fragment, and under different conditions – strongly suggesting that the segment complementary to adjacent viral DNA sequence
• The structure of hexon mRNA and EcoRI DNA hybrids suggest three short sequences are spliced into the 5’ tail of hexon mRNA during post transcription processing.
• These RNA sequences originate upstream from those coding the body of hexon.
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An Amazing Sequence Arrangement at the 5’ Ends of Adenovirus 2 Messenger RNA
Louise T. Chow, Richard E. Gelinas, Thomas R. Broker and Richard J. Roberts
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Experiments• Many techniques are the same (R-
loop, restriction mapping) but instead mixed late RNA is used
• The hybridizations are subsequently probed with restriction fragments from the r strand and other parts of the genome to map unhybridized fragments
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Observations & Conclusions
• Sequences present at three separated sites on the R-strand of the Ad2 genome are complementary to a continuous sequences at the 5’ end of late Ad2 mRNAs
• Additional late mRNAs show different headers from non-adjacent DNA sequence
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. . . continued
• These observations are inconsistent with any previous mechanism proposed for eukaryotic mRNA synthesis
• Together both papers present data that helped develop understand of eukaryotic mRNA processing, the presence of introns, and splicing.
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Questions ?