James Darnell explains how chemical signals turn eukaryotic genes on and off.

Hi, I'm James Darnell. I'm interested in how chemical signals turn eukaryotic genes off and on. Multicellular organisms respond to changes in their environment just like single-cell organisms. Cells in multicellular organisms also need to "talk" to one another to coordinate growth and development. Cells communicate with chemical signals. Receptors intercept the signals and pass the messages to a series of intracellular molecules. Messages that carry instructions for protein synthesis are relayed to the nucleus and its DNA. We call this relay "signal transduction," because the message passes through many different forms during its journey. Interferon is one of many signals used by animal cells for communication. Cells infected by RNA viruses release the chemical to a receptor on a nearby uninfected cell. Interferon instructs the uninfected cell to make defensive proteins in preparation for the viral attack. Interferon is one of many signals used by animal cells for communication. Cells infected by RNA viruses release the chemical to a receptor on a nearby uninfected cell. Interferon instructs the uninfected cell to make defensive proteins in preparation for the viral attack. We knew how this pathway was initiated. First, a receptor on an uninfected cell binds an interferon molecule. The interferon receptor has two subunits. When interferon binds, the subunits come together. This activates two molecules called JAKs (Janus kinases), which are attached to the subunits. Activation occurs through phosphorylation. JAKs and other enzymes are converted to their active states when a phosphate group attaches to a particular amino acid. The phosphate changes the enzyme's shape, thereby changing its activity. At this point, we lost track of the pathway. We looked for other molecules that carry the message into the nucleus. Because interferon causes synthesis of specific proteins, the final molecule in the pathway must attach to the cell's DNA and activate transcription. We decided to look for this molecule first. Transcriptional activators normally bind to regulatory regions, called enhancers, located upstream, or 5', of the genes they turn on. We found the transcriptional activator in the interferon pathway by identifying the DNA sequence of the enhancer. First, we identified a gene that is turned on by interferon and then we isolated a DNA fragment upstream of this gene. We made many copies of this fragment and radiolabeled them at one end. Then we mixed the enhancer fragments with nuclear proteins extracted from interferon-treated cells. The activator was in this extract, though we still didn't know which one it was. For comparison, we also mixed the enhancer fragments with extract from untreated cells. No activator was present in this extract. During incubation, the transcription activators from the interferon-treated cells bound to the enhancer fragments. Then, we added a small amount of DNase to each container. At low concentrations, DNase cuts each strand only once, and each strand is cut in a random location. Regions of DNA with bound proteins, however, were protected from cutting. In the extract from the untreated cells, there were no bound proteins, and DNase cut everywhere. The result was a collection of fragments of every possible length. We ran each solution out on an acrylamide gel — the same kind of gel used in DNA sequencing. The pieces segregate by size: largest at the top, smallest at the bottom. Let's look at the untreated extract first. We had previously sequenced the region so we knew which band corresponded to which nucleotide. Each band represents one fragment size. Because all possible fragment sizes are present, each band also represents a nucleotide in the enhancer. In the interferon-treated extract, some of the bands are missing. These are the bases that were protected by the activators. We simply read the sequence inside the hole — T C A C T T T — to reveal the binding region's sequence. This is a "footprint" of the activator binding site. Remember, we were trying to identify the protein that activates transcription. Now with its binding sequence in hand, we made another radioactive probe to fish it out. Again, we incubated the probe with nuclear extracts from interferon-treated and untreated cells. As before, the probe attached to a protein complex that existed in the interferon extract. We isolated the proteins that bound to the probe and discovered that they have two jobs! Not only do they activate transcription in the nucleus, but they also carry the signal from the receptor to the nucleus. Let's go back to the receptor. Remember, interferon-binding activates the JAKs. In turn, the JAKs activate two of the three proteins we found by footprinting. After activation, these molecules, called STATs (Signal Transducers and Activators of Transcription), combine with the third molecule called p48. The entire complex moves into the nucleus, binds to the enhancer, and turns on gene transcription. The enzyme produced from this gene is called 2’,5’ ­ oligoadenylate synthetase (2-5 A). When a retrovirus enters the cell, the double-stranded viral RNA helps activate 2-5A. This leads to activation of an RNase through the dimerization of its two components. The RNase dimer degrades the viral RNA. The interferon pathway is the simplest example of eukaryotic gene activation found so far. Other eukaryotic pathways, initiated by hormones and growth factors, are less direct and use many more intermediates. Any defect in these pathways can lead to aberrant cell responses or growth. Mutations in some pathway molecules lead to unchecked cell growth and cancer. To do this we isolated one of the many mRNAs that the cell produces after treatment with interferon, and made a copy of it using reverse transcriptase. The DNA copy (cDNA) of the mRNA was labelled with radioactivity and used to probe a phage library. We used this cDNA probe to find its gene in a genomic library — a collection of phage that contain various bits of human DNA. We plate the phage library onto agar plates covered with a "lawn" of bacteria. When a phage infects a bacteria, it multiplies and kills the cell. Phage progeny infect the surrounding bacteria and we see a visible hole in the bacterial lawn. Many holes — called plaques — develop on the plate. Each represents a different phage. Then we pressed a piece of filter paper against the plate to transfer phage to the filter. We can incubate this filter with our radioactive cDNA probe. We put the filter in a plastic bag and add the radioactive cDNA. The probe will hybridize to phage that carries the gene. After rinsing the excess probe off the filter, we exposed the filter to X-ray film. And we can go back and isolate the phage that has "our" gene. Having found the gene, we can now examine its regulatory regions. There must be a binding site for the transcription activator that will turn on this gene. The activator site usually lie 5', upstream, of the gene. We searched a 177 base pair region with a technique called DNA footprinting.

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