Concept


Growth and development require that cells communicate with each other and react to signals that come from other parts of the body. Notably, hormones, released by various glands, travel throughout the body to stimulate the growth of certain cell types. Cells capable of being stimulated by a particular hormone possess a specific receptor anchored in the cell membrane. The binding of a hormone to its receptor initiates a series of molecular transformations, called signal transduction, that relay the growth signal through the cell. First, the receptor transduces the signal through the cell membrane to the internal membrane surface, where it activates protein "messengers." These messengers are part of and inititate a cascade of chemical reactions, often involving the addition of phosphate groups. This is the signal that passes through the cytoplasm and into the nucleus. In the final step of signal transduction, DNA-binding proteins attach to regulatory sequences and start DNA replication or transcription.

Animation


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. 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.

Gallery


Audio/Video


Video Interviews

James E. Darnell, Jr.

Dr. James Darnell is a Vincent Astor Professor of Molecular Cell Biology at Rockefeller University in New York City.

Clip 1 (0:55)
Can eukaryotic cells react quickly to environmental changes?

Clip 2 (0:38)
cAMP pathways vs protein pathways

Clip 3 (1:29)
What does phosphate do in the pathway?

Clip 4 (1:29)
What's the difference between tyrosine and serine phosphorylation?

Clip 5 (0:40)
Tyrosine phosphorylation and cancer

Clip 6 (0:57)
Other signaling pathways

Biography


 

JAMES E. DARNELL, JR. (1930-)

Jim Darnell was born in Columbus, Mississippi - an impoverished region near the Alabama border - on September 9, 1930. His mother was a dietician, and young Jim saw medicine as a way to get out of the South. He raced through college at the University of Mississippi in three years, then worked at the local Air Force base for nine months to raise money for medical school. In 1951, he started studying for his M.D. at Washington University in St. Louis.

Because science news was not well reported in the '40s and '50s, Darnell got his first exposure to lab work in medical school. He was drawn to microbiology; it was a subject where investigators had some control over their subjects. He tried to infect mice with a strain of streptococcus in his first experiments but failed. He had better success infecting rabbits and wrote up the results in his first set of papers. At the time, streptococcus infections produced lesions on the heart, but this disease virtually disappeared after the discovery of penicillin.

After receiving his medical degree and interning for a year, Darnell went to Harry Eagle's lab at NIH under a M.D.-to-scientist training program. Eagle was developing ways to culture human and mouse cells and set Jim to work on studying the biochemistry of animal viruses. Knowing next to nothing about animal viruses, Darnell was fortunate to share his lab bench with Salvador Luria's third graduate student who taught him everything he needed to know to study the polio virus.

After a brief sojourn in Paris to work in François Jacob's lab, Darnell worked at MIT, the Albert Einstein College of Medicine, and Rockefeller University on the relationship between mRNA and hnRNA. hnRNA was believed to be the precursor to mRNA, and despite making some key discoveries, Darnell admits that he could not free his imagination from the idea of colinearity and envision an hnRNA spliced to produce a smaller mRNA.

At this time, Darnell turned his attention to the question he had pondered since Paris: how were genes regulated in animal cells? This led to the discovery of the STAT and the Jak-STAT pathway of transcription control.

In his spare time, Jim Darnell is an avid tennis player and, despite serving on the Cold Spring Harbor Board of Trustees, has never played Cold Spring Harbor's most famous tennis player, Jim Watson. In his early days, he played the clarinet and he still enjoys listening to music. He and his wife, Jane, have three sons: the oldest working in money management, the middle in science, and the youngest, inheriting his left-wing tradition, in public interest research.

Factoid

Links


 

Links

Cell Communication: The Inside Story

How do all the molecules in a signaling pathway find each other in the cytoplasm? How come signals don't get crossed? Find out in the article from Scientific American.

Mechanism of Action: Hormones with Cell Surface Receptors

We've spared you from the cAMP second messenger system in this concept. Scroll past the introduction to receptors and get a short description of it here.

Bibliography

  • Darnell Jr., J.E., Kerr, I.M., Stark, G.R., 1994, Jak-STAT pathways and Transcriptional Activation in Response to IFNs and Other Extracellular Signaling Proteins, Science, 264: 1415-1421.

  • Leaman, D.W., 1998, Mechanisms of Interferon Action, Progress in Molecular and Subcellular Biology, 20, 101-142.

  • Levy, D., Larner, A., Chaudhuri, A., Babiss, L.E., and Darnell Jr., J.E., 1986, Interferon-Stimulated Transcription: Isolation of an Inducible Gene and Identification of its Regulator Region, Proc. Natl. Acad. Sci., U.S.A., 83: 8929-8933.

  • Levy, D.E., Kessler, D.S., Pine, R., Reich, N., and Darnell Jr., J.E., 1988, Interferon-Induced Nuclear Factors That Bind a Shared Promotor Element Correlate with Positive and Negative Transcriptional Control, Genes and Development, 2: 383-393.

  • Levy, D.E., Kessler, D.S., Pine, R., Reich, N., and Darnell Jr., J.E., 1989, Cytoplasmic activation of ISGF3, the positive regulator of interferon-alpha-stimulated transcription, reconstituted in vitro, Genes and Development, 3: 1362-1371.

  • Stark, G.R., Kerr, I.M., Williams, B.R.G., Silverman, R.H., Schreiber, R.D., 1998, How Cells Respond to Interferons, Annu. Rev. Biochem., 67: 227-264.

Glossary



Children resemble their parents.
Genes come in pairs.
Genes don't blend.
Some genes are dominant.
Genetic inheritance follows rules.
Genes are real things.
All cells arise from pre-existing cells.
Sex cells have one set of chromosomes; body cells have two.
Specialized chromosomes determine gender.
Chromosomes carry genes.
Genes get shuffled when chromosomes exchange pieces.
Evolution begins with the inheritance of gene variation.
Mendelian laws apply to human beings.
Mendelian genetics cannot fully explain human health and behavior.
DNA and proteins are the molecules of the cell nucleus.
One gene makes one protein.
A gene is made of DNA.
Bacteria and viruses have DNA too.
The DNA molecule is shaped like a twisted ladder.
A half DNA ladder is a template for copying the whole.
RNA is an intermediary between DNA and protein.
DNA words are three letters long.
A gene is a discrete sequence of DNA nucleotides.
The RNA message is sometimes edited.
Some viruses store genetic information in RNA.
RNA was the first genetic molecule.
Mutations are changes in genetic information.
Some types of mutations are automatically repaired.
A chromosome is a package for DNA.
Higher cells incorporate an ancient chromosome.
Some DNA does not encode protein.
Some DNA can jump.
Genes can be turned on and off.
Genes can be moved between species.
Different genes are active in different kinds of cells.
Master genes control basic body plans.
Development balances cell growth and death.
A genome is an entire set of genes.
Living things share common genes.
DNA is only the starting point for understanding human biology.
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