Figure 10.8 Demonstrating the Existence of Introns
In 1977, two independent research groups (Susan Berget, Claire Moore, and Phillip Sharp; and Louise Chow, Richard Gelinas, Tom Broker, and Richard Roberts) working with adenovirus mRNA discovered that eukaryotic DNA was not contiguous, but rather contained introns, or noncoding regions, that are spliced out of mRNA prior to protein translation. The researchers used nucleic acid hybridization to demonstrate this surprising occurrence. Specifically, the β-globin gene was denatured to separate the strands of DNA and incubated with a probe consisting of β-globin mRNA from mature mRNA transcripts of exons 1 and 2. The probe hybridized with the genomic DNA and caused the DNA to loop out in the region where the noncoding sequence was displaced by the mRNA. This result indicated the presence of an intron, or an intervening DNA sequence, between exons 1 and 2. This method has been used to determine the intron–exon structure for a number of other genes as well. For each intron, one can expect the mRNA to force the DNA into a loop. For example, if there were three exons and two introns, two loops would be visible by this hybridization method. The discovery of introns radically changed the way scientists viewed eukaryotic gene mRNA. In 1993, Phillip Sharp shared the Nobel Prize in Physiology or Medicine with Richard Roberts “for their discovery of split genes.” Early on, many researchers believed that introns were merely “junk DNA” with no particular function. However, more recent studies suggest introns function in a number of cellular processes, including catalytic activity during mRNA splicing, gene regulation, and exon-shuffling, which is thought to play a role in evolution.
Original Papers
Berget, S. M., C. Moore, and P. A. Sharp. 1977. Spliced segments at the 5' terminus of adenovirus 2 late mRNA. Proceedings of the National Academy of Sciences 74: 3171–3175.
http://www.pnas.org/content/74/8/3171.full.pdf+html
http://www.ncbi.nlm.nih.gov/pubmed/269380?dopt=Abstract
Chow, L. T., R. E. Gelinas, T. R. Broker, and R. J. Roberts. 1977. An amazing sequence arrangement at the 5' ends of adenovirus 2 messenger RNA. Cell 12: 1–8.
http://www.ncbi.nlm.nih.gov/pubmed/902310
Links
Clancy, S. 2008. RNA Splicing: Introns, Exons and Spliceosome. Nature Education 1(1)
http://www.nature.com/scitable/topicpage/RNA-Splicing-Introns-Exons-and-Spliceosome-12375
The Cell: A Molecular Approach: The Complexity of Eukaryotic Genomes
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.box.614
National Human Genome Research Institute: 1977: Introns Discovered
http://www.genome.gov/25520306
North Dakota State University: Cloning and Molecular Analysis of Genes: Exons and Introns
http://www.ndsu.nodak.edu/instruct/mcclean/plsc431/cloning/clone8.htm
Scientific American: What is known about the function of introns, the nonencoding sequences in genes?
http://www.scientificamerican.com/article.cfm?id=what-is-known-about-the-f
Drs. Richard Roberts and Phillip Sharp: The Nobel Prize in Physiology or Medicine 1993
http://nobelprize.org/nobel_prizes/medicine/laureates/1993/
MIT: Sharp lab home page
http://web.mit.edu/sharplab/home.html
New England Biolabs: Dr. Richard Roberts
http://www.northeastern.edu/cos/faculty/sir-richard-john-roberts/
Figure 10.10 Deciphering the Genetic Code
In 1961, Nirenberg and Matthaei cracked the genetic code by using an artificial mRNA in which all of the bases were uracil (poly U). In this experiment, the scientists prepared a bacterial extract that contained all of the components needed to translate proteins and then added the mRNA homopolymer. Results showed that the cell-free extract produced a polypeptide composed entirely of the amino acid phenylalanine. Similarly, Nirenberg and Matthaei went on to test poly A and poly C, which produced lysine and proline, respectively. Although at the time the pair did not test poly G due to technical difficulties with mRNA synthesis, this homopolymer would have resulted in the production of glycine. This experiment, commonly referred to as “the poly U experiment,” laid the foundation for Nirenberg’s groundbreaking work on the genetic code. He continued working on the code until he had completely deciphered it in 1966. Two years later, Nirenberg shared the Nobel Prize in Physiology or Medicine 1968 with two other scientists “for their interpretation of the genetic code and its function in protein synthesis.”
Original Papers
Nirenberg, M. W., and J. H. Matthaei. 1961. The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proceedings of the National Academy of Sciences 47: 1588–1602.
http://www.pnas.org/content/47/10/1588.full.pdf+html
Nirenberg, M. W., J. H. Matthaei, and O. W. Jones Jr. 1962. An intermediate in the biosynthesis of polyphenylalanine directed by synthetic template RNA. Proceedings of the National Academy of Sciences 48: 104–109.
http://www.pnas.org/content/48/1/104.full.pdf+html
Matthaei, J. H., O. W. Jones Jr., R. G. Martin, and M. W. Nirenberg. 1962. Characteristics and composition of RNA coding units. Proceedings of the National Academy of Sciences 48: 666–677.
http://www.pnas.org/content/48/4/666.full.pdf+html
Jones, O.W. Jr., and M. W. Nirenberg. 1962. Qualitative survey of RNA codewords. Proceedings of the National Academy of Sciences 48: 2115–2123.
http://www.pnas.org/content/48/12/2115.full.pdf+html
Nirenberg, M. W., J. H. Matthaei, O. W. Jones Jr., R. G. Martin, and S. H. Barondes. 1963. Approximation of Genetic Code Via Cell-Free Protein Synthesis Directed by Template RNA. Federation Proceedings 22: 55–61.
http://profiles.nlm.nih.gov/JJ/B/B/J/J/
Nirenberg, M. W., P. Leder, M. Bernfield, R. Brimacombe, J. Trupin, F. Rottman, and C. O'Neal. 1965. RNA codewords and protein synthesis, VII. On the general nature of the RNA code. Proceedings of the National Academy of Sciences 53: 1161–1168.
http://www.pnas.org/content/53/5/1161.full.pdf+html
Links
Smith, A. 2008. Nucleic Acids to Amino Acids: DNA Specifies Protein. Nature Education 1(1)
http://www.nature.com/scitable/topicpage/Nucleic-Acids-to-Amino-Acids-DNA-Specifies-935
Ralston, A., and K. Shaw. 2008. Reading the Genetic Code. Nature Education 1(1)
http://www.nature.com/scitable/topicpage/Reading-the-Genetic-Code-1042
Dr. Marshall Nirenberg: The Nobel Prize in Physiology or Medicine 1968
http://nobelprize.org/nobel_prizes/medicine/laureates/1968/
Lasker Foundation: An Interview with Marshall Nirenberg
http://www.laskerfoundation.org/media/v_nirenberg.htm
Genome News Network: Marshall Nirenberg (1927–2010) Cracks the Genetic Code
http://www.genomenewsnetwork.org/resources/timeline/1961_Nirenberg.php
Office of NIH History: The Poly-U Experiment
http://history.nih.gov/exhibits/nirenberg/HS4_polyU.htm
National Library of Medicine: Profiles in Science: The Marshall W. Nirenberg Papers: Synthetic RNA and the Poly-U Experiments, 1957–1962
http://profiles.nlm.nih.gov/JJ/Views/Exhibit/narrative/syntheticrna.html
National Library of Medicine: Profiles in Science: The Marshall W. Nirenberg Papers: Translating the Code of Life and the Nobel Prize, 1962–1968
http://profiles.nlm.nih.gov/JJ/Views/Exhibit/narrative/codeoflife.html
Wall Street Journal: Scientist Set Stage for Genetic Engineering
http://online.wsj.com/article/SB10001424052748703626604575011450122739496.html
Figure 10.20 Testing the Signal
For proper organelle localization, intracellular signaling is required, directing polypeptides to the correct destination. Often such signaling involves a localization signal or tag, which is a specific amino acid sequence that interacts with a receptor protein on the surface of the organelle that is the correct “destination.” Dingwall and colleagues investigated the nuclear localization signal used by the nuclear protein nucleoplasmin by experimentally manipulating both this nuclear protein, as well as the cytoplasmic protein pyruvate kinase. The researchers first determined that a specific “tail” fragment of amino acids, not the main core part of nucleoplasmin, was both necessary and sufficient for localization from the cytoplasm to the nucleus. This was experimentally determined in monkey cells by injecting nucleoplasmin with and without the “tail” and observing localization using immunofluorescence microscopy. The nuclear localization function of this “tail” was then tested by injecting into cells the normal cytoplasmic protein pyruvate kinase, as well as pyruvate kinase linked to the nucleoplasmin “tail.” As predicted, the normal pyruvate kinase remained in the cytoplasm, whereas the pyruvate kinase linked to the nucleoplasmin “tail” entered the nucleus. Intracellular trafficking is important for many polypeptides, including those destined for mitochondria or chloroplasts. To study protein import into chloroplasts, Schnell and colleagues used immunofluorescence microscopy and synthetic peptides, determining that the signal polypeptides bind to specific receptor proteins on the chloroplast surface. Recently, defects in protein localization have been implicated in several human diseases. For example, inclusion-cell disease is due to a lack of the necessary localization signal for transport of certain proteins from the Golgi apparatus into the lysosomes, and Zellweger syndrome results from a mutated receptor protein, inhibiting proteins from entering the peroxisome.
Original Paper
Dingwall, C., J. Robbins, S. M. Dilworth, B. Roberts, and W. D. Richardson. 1988. The Nucleoplasmin Nuclear Location Sequence is Larger and More Complex than That of SV-40 Large T Antigen. The Journal of Cell Biology 107: 841–849.
http://jcb.rupress.org/cgi/reprint/107/3/841
Links
Schnell, D. J., G. Blobel, and D. Pain. 1991. Signal peptide analogs derived from two chloroplast precursors interact with the signal recognition system of the chloroplast envelope. Journal of Biological Chemistry 266: 3335–3342.
http://www.jbc.org/content/266/5/3335.abstract
Wikipedia: Nuclear localization signal
http://en.wikipedia.org/wiki/Nuclear_localization_signal
University College London: K. Singh Dulai: Signal peptides
http://www.cryst.bbk.ac.uk/pps97/assignments/projects/dulai/signal.html#SP
Kimball’s Biology Pages: Protein Kinesis: Getting Proteins to Their Destination
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/ProteinKinesis.html
Wikipedia: Inclusion-cell disease
http://en.wikipedia.org/wiki/I-cell_disease
NCBI: Genes and Disease: Zellweger syndrome
http://www.ncbi.nlm.nih.gov/books/NBK22240/
University of Utah: Genetic Science Learning Center: Amazing Cells
http://learn.genetics.utah.edu/content/begin/cells/