Concept

All living organisms store genetic information using the same molecules — DNA and RNA. Written in the genetic code of these molecules is compelling evidence of the shared ancestry of all living things. Evolution of higher life forms demanded the development of new genes to support different body plans and types of nutrition. Even so, complex organisms retain many genes that govern core metabolic functions carried over from their primitive past. DNA accumulates mutations over evolutionary time; the number of sequence differences between the DNA of two organisms provides a measure of their relatedness. Because of redundancies in the genetic code, point mutations in a gene often do not change the amino acid encoded. Also, amino acid substitutions can be tolerated in nonessential regions which do not directly participate in chemical interactions of the protein. Even though a gene may have accumulated many such mutations, one or several conserved regions may show its likeness (homology) to genes of other organisms.

Animation

Hi, I’m Michael Wigler. Organisms share similar genes because they have inherited them from common ancestors. Even humans and yeast share similar genes! I was interested in genes involved in cancer, like the human ras gene. This gene contributes to uncontrolled cell growth and proliferation when mutated, for example, by the hydrocarbons in cigarette smoke. ras does not exist to cause cancer. We wanted to find out what it normally does so we looked for the same gene, or homolog, in yeast. If yeast have ras, we could use yeast instead of people to study the gene’s normal role. Nowadays, we can simply search for homologs on a large computer database, but in the early 1980s, we searched for homologs using DNA hybridization. Radioactive fragments of the human ras gene were used as probes to screen a gene library containing the entire yeast genome. We isolated clones that bound to the probe. Each of these clones contained a portion of the same gene — the yeast's ras gene. We sequenced the gene, deduced its amino acid sequence, and compared it to the human sequence. The yellow shading highlights amino acids that are identical in both proteins. As you can see, the two protein sequences are extremely similar. This means the gene has been conserved during the billion years since yeast and humans shared a common ancestor. All other eukaryotes also have this gene, and all the proteins resemble each other. The protein sequence has been conserved because it is essential for basic cell processes in these organisms. In fact, 33% of the yeast's genes are conserved in our own genome. When we compare the nucleotide sequences of the genes we also see similarity, but not as much. This is because the genetic code is redundant. Most amino acids are encoded by two to six different codons, so a change in one nucleotide does not necessarily change the amino acid. In the sequence below, 18 out of the 21 amino acid matches are encoded by different codons. Though we saw that the two genes are structural homologs — their amino acid sequences are very similar — that does not mean they do the same job. To see if the two genes perform the same function, we inserted the human gene into yeast cells. We started with a strain of yeast that was missing ras. The strain also lacked the leu gene, so we needed to supplement the medium with the amino acid leucine for the cells to grow. Then we added a plasmid to the yeast culture. The plasmid contained a functioning leu gene and a human ras gene that was under the control of a galactose promoter. We cut the plasmid and added it to the yeast culture. The plasmid integrated into the DNA of a few yeast cells. We isolated cells that had integrated the plasmid by spreading the yeast culture on a medium lacking leucine. Yeast that incorporated the plasmid and its leu gene survive and reproduce on this medium. Then we starved the transformed yeast so they would produce spores. Each yeast cell produces four spores encased in a capsule. We separated the spores onto different culture plates and watched them for germination. We knew from prior experience that yeast spores without ras would not germinate. None of the spores germinated on the glucose medium, because the galactose promoter needs galactose to turn on the human ras gene. On the galactose medium, the human ras gene was transcribed, and these spores germinated! Let's take a closer look to see why human ras can substitute for yeast ras and rescue the mutant yeast. (The amino acids are only labeled in the first row; the remaining residues are represented by dashes.) The red arrows point to a region of very high similarity in the first 80 residues. Here, the two proteins share 90% of their amino acids. Part of this region binds GTP during signal transduction, a job performed by all ras proteins – from yeast to fruit flies to humans. The GTP-binding domain has been conserved, because many substitutions here result in structural changes. Substituting the glycine at position 12, for example, pushes an adjacent branch away from the binding region. Click the hand to see the change. This mutation prevents the hydrolysis of GTP into GDP, and the protein activity can't turn off. In other words, the mutation turns a normal human ras gene into an oncogene — a gene that can cause uncontrolled cellular growth and cancer. 20% of human cancers carry this mutated ras. Other positions in this domain tolerate some change, as long as the substituted amino acid is similar. In yeast, position 11 is filled by glycine, the smallest amino acid, while in humans it is filled by alanine, the next smallest residue. There is no change in the protein's structure. We also noticed highly variable regions of the proteins. Some of these are not important for the protein's function. For example, the beginning of the ras proteins not only vary in the type of amino acid but also in the number of residues. Humans have three residues at the beginning of the protein, while yeast have ten! In human ras, amino acids in these positions can be replaced with unrelated residues without affecting the protein’s function. Hi, I’m Harold Varmus, and I’m Michael Bishop. Conserved genes tell the story of evolution as Darwin envisioned it: species inherit traits, with modification, from ancestral species. But genes are not always passed on in a linear fashion — organisms sometimes steal genes from one another. When we were studying cancer-causing retroviruses, we discovered that a virus had stolen a gene from a chicken! Remember, retroviruses carry their genetic information in RNA. A typical retrovirus has only three genes. A cancer-causing retrovirus has an additional gene. We started with two avian sarcoma viruses (ASV): the wild-type ASV that causes cancer in chickens and a mutant ASV that was missing the cancer-causing gene (src). Our first step was to isolate a probe for src. Using reverse transcriptase, we made single-stranded cDNAs from the wild-type virus and labeled them with radioactive hydrogen. We combined the cDNAs with single-stranded RNA we isolated from the mutant virus. Complementary sequences hybridized with each other to produce double-stranded molecules. Because the mutant virus lacked the src gene, the src cDNA was left in its single-stranded state. The single-stranded src cDNA we isolated became our probe. First, we combined the probe with single-stranded chicken DNA and let any complementary strands hybridize. We removed the single-stranded DNA with S1 nuclease, which only digest single-stranded DNA, and were left with radioactive hybrids. The probe had bound to the chicken's own src gene! But which came first, the chicken src or the viral src? We used the same probe to look for src genes in other animals. Birds, humans, mice and salmon had the gene, but invertebrates and bacteria did not. Further analysis showed that the viral src hybridized most completely with the chicken gene. It seemed unlikely that the virus gave src to all these animals when its src is most similar to the chicken's. We concluded the virus stole the gene from the chicken. Further proof came when we looked at the structure of the viral and chicken src genes. Hybridizing the viral src DNA to the chicken src revealed loops of introns in the chicken gene. The chicken src has introns, just like most other eukaryotic genes. The virus got rid of these introns when it captured the chicken gene in a process called transduction. The process depends on two viruses integrating their genomes into two places in the host. Sometimes, one of the viruses integrates next to an oncogene like src. If the downstream LTR – a sequence needed for viral integration – is deleted, the oncogene is copied into the viral progeny when the DNA is transcribed. The introns in the src gene are spliced out. Meanwhile, the RNA polymerase transcribes the second provirus. This normal virus RNA produces the viral capsid. Both RNAs get packaged into a capsid, where recombination generates a viral genome with both of the required LTRs; gag, env, and pol; and the src gene. This virus causes cancer when it infects a host, because the src gene, normally expressed at low levels, is controlled by a strong virus promoter. The src protein is overproduced, leading to uncontrolled growth.

Gallery

A young Mike Wigler with pet snake.

Mike Wigler mugging for the camera.

Mike Wigler (R) with his older brother and pet dog.

Harold Varmus and Michael Bishop at a 1978 Cold Spring Harbor meeting.

Harold Varmus, President and Chief Executive Officer of Memorial Sloan-Kettering Cancer Center.

A very young J. Michael Bishop.

J. Michael Bishop as a high school graduate.

J. Michael Bishop as a graduate student.

J. Michael Bishop, pitcher of his softball team.

Audio/Video

Audio Glossary

Mutation, Oncogene

Video Interviews

Michael Wigler

Dr. Mike Wigler is a Professor in the Watson School of Biological Science at Cold Spring Spring Harbor Laboratory.

Clip 1 (0:56)
Why look for a cancer gene in yeast?

Clip 2 (0:57)
Cancer genes are fundamental for cell survival.

Clip 3 (0:36)
Looking for the function of ras in frog eggs.

Clip 4 (1:16)
What is the function of ras?

Clip 5 (0:57)
What's a homolog?

Clip 6 (0:30)
What happens to the ras protein when the gene is mutated?

Biography

Mike Wigler and his group were among the first to clone and characterize human oncogenes. Mike Bishop and Harold Varmus worked out how retroviruses transform normal cells to cancerous ones.

MICHAEL WIGLER (1947-)

Boredom with the suburbs may have been responsible for Mike Wigler's career as a scientist. Before his family moved from the Bronx, he loved to play baseball with friends or capture snakes in the park.

"I was very unhappy leaving the city," Wigler recalled, "because there are a lot of parks in the city and kids play in them. We came out to the suburbs, where theoretically there's more nature, but there were very few parks and kids didn't play outside."

Wigler channeled his boredom into his studies and he began studying chemistry in the fifth grade. "The precipitating event was I wanted to build a rocket ship, and I needed to know about fuel and oxidation/reduction. Basically, I wanted to make explosions."

His plan was thwarted by his father, a high school chemistry teacher, who refused to bring home anything dangerous. Wigler's interest in chemistry shifted to math and physics before college and he went off to Princeton to major in mathematics.

At Princeton, Wigler excelled in his classes and he started enrolling in graduate level classes during his sophomore year. During a leave of absence he spent at his parents' house, Wigler decided to leave mathematics and go to medical school to devote his life to helping people.

"Math has very little social relevance," Wigler explained in Natural Obsessions, a book about the search for cancer genes. "In the long run it's useful to society, but in the short run math is a more autistic activity. I wanted to do something with my life that might be socially useful."

Asked why he didn't finish medical school at Rutgers, Wigler joyously replied "because I flunked out!" But in reality Wigler's mind was wandering again. Instead of planning explosions, he played tournament chess. Eventually he was given another leave of absence.

Perusing the New York Times "help wanted" section, Wigler found an ad for a lab technician at Columbia University with Bernie Weinstein. "I wasn't what he was looking for, but he created a position and hired me part-time. I was there to play, I guess, until I figured out what I wanted to do with my life."

Weinstein was interested in cancer and studied chemical carcinogens that altered DNA. Trained in mathematics, and imbued with the arrogance that those in the profession have toward biologists, Wigler thought the approach was futile. "It was clear to me that if things that caused cancer were mutagens, then cancer was a disease of mutation. To solve cancer - which seemed to me to be an interesting problem to work on - one would have to develop genetic tools."

Wigler began to develop these genetic tools as a graduate student under Richard Axel at Columbia. Wigler looked at how geneticists succeeded in transferring genes from one bacterium to another, and helped develop a system to do the same thing with mammalian cells.

After he moved to Cold Spring Harbor Laboratory in 1979, Wigler's lab and others used this system to insert a human cancer gene into normal human cells. The normal cells turned cancerous, and the first human oncogenes were discovered.

Wigler's lab still searches for new human oncogenes and studies the function of the genes in yeast and mammalian cells. When Wigler is not in the lab, he plays the piano - with no rhythm - or tries his hand at bridge.

JOHN MICHAEL BISHOP (1936-)

Perhaps one of the misconceptions about Nobel Prize-winning scientists is that they emerge from the womb ready to peer under a microscope and discover the secrets of life. Not so for J. Michael Bishop whose first passion was music. Bishop learned to sing and play the piano in the two small country churches his father ministered in York, Pennsylvania.

Bishop's second passion was history, sparked by an engaging teacher in his two-room schoolhouse. He did well in school, but did not encounter science until high school when he befriended the local doctor. The doctor took him to see patients and witness surgery, so Bishop decided to prepare for medical school when he entered Gettysburg College in 1953.

Though he graduated from college with a degree in chemistry, other subjects tried to pull Bishop away from medical school. History, philosophy, and writing all had their chances, but the only thing Bishop felt strongly about was pursuing an academic career, even though he wasn't sure what subject he wanted to teach. With that in mind, his college advisors steered him to Harvard Medical School.

Boston's supply of symphonies and art museums provided enough distractions for a small-town boy in his first experience in urban living. Medical school, however, was not the teacher-training haven he expected. Instead, it became clear that the path to an academic career led through research, not teaching, and Bishop had no research experience. He sought summer employment in a neurobiology lab, but was rejected.

Two pathologists at Massachusetts General Hospital rescued Bishop and introduced him to research. By his third year, he was drawn to animal virology, partly because the field was still open to newcomers, and he moved to another lab. There he "learned the inebriation of research, the practice of rigor, and the art of disappointment."

Further training through the National Institute of Health's Research Associate Training Program solidified Bishop's research skills, and he published his first papers on the replication of the polio virus. In 1968, he accepted a faculty position at the University of California, San Francisco, where he began working on the replication of retroviruses.

In 1970, Harold Varmus joined the lab as a post-doc and changed Bishop's life. Together, they decided to solve the mystery of how the Avian (or Rous) Sarcoma Virus transformed normal cells into cancers. The research revolutionized people's thinking about cancer - they now realized that the cell's own genes were responsible - and it garnered the two the Nobel Prize in Physiology or Medicine in 1989.

Though best known for his research, Bishop is a popular teacher at UCSF and has twice won the school's Excellence in Teaching award. In addition, he worked on the university's outreach program to promote science education in public schools. He says, "Science is the transcendant cultural force of our time. No one can consider themself in tune without time or our future without a general knowledge of science. It is also the primer approach to discovery and learning, so its principles can be useful to almost anyone."

Since 1998, J. Michael Bishop has been Chancellor of UCSF. He lives in San Francisco, California with his wife, Kathryn Ione Putman. They have two sons, Dylan Michael Dwight and Eliot John Putnam. If reincarnated, Dr. Bishop wishes to become an exceptionally talented musician in a string quartet.

HAROLD ELIOT VARMUS (1939-)

Midway through his first year as a graduate student in English Literature at Harvard, Harold Varmus had a dream that terrified him. He was an English professor - the job he was training for - but missed a day of lecturing due to illness. His students were enthralled with the news that there would be no class. Upon waking, Varmus thought that if he were a doctor, no one would be happy if he didn't show up for work. And with that thought, Varmus redirected his curiosity first to medicine, then to science, and finally to running the largest biomedical institution in the world, the National Institutes of Health.

Varmus originally planned on becoming a doctor like his father as he grew up on the South Shore of Long Island. He enjoyed the outdoors - fishing in the summer and skiing in the winter - but was inept on the football and baseball fields. He turned to reading when he attended the local public schools that were dominated by team sports.

In 1957, Varmus began pre-med studies at Amherst College but was seduced by the academic life. He drifted from science to English literature, got involved in politics, and ran the college newspaper. After completing his senior thesis on Charles Dickens, he packed up for graduate school at Harvard with a Wilson fellowship in hand.

After leaving graduate school, Varmus studied medicine at Columbia College of Physicians and Surgeons. Initially attracted by practicing medicine abroad, an apprenticeship in a mission hospital in Bareilly, India tempered this desire, and he switched to basic medical research. He first experienced life in the lab as a Clinical Associate at NIH studying gene regulation in bacteria.

A year later in 1970, Varmus went to the University of California, San Francisco to study tumor viruses with Mike Bishop. At the time, many scientists thought that these viruses caused cancer by injecting their genes into the host's own genome. Bolstering this view, viral genes from the tumor viruses were found in infected animals. But Varmus and Bishop found that these viral genes had been stolen from the animals in the first place. The genes that caused cancer came from within - they were simply damaged. For this work, Varmus and Bishop shared the Nobel Prize for Physiology or Medicine in 1989.

Varmus stayed at UCSF until 1993 when he left to run the National Institutes of Health. Though his friends thought he didn't have the patience for the job - and he had no administrative experience outside of his own lab - Varmus stroked the egos of Congressmen of both parties enough to increase the NIH's budget from 11 billion to 16 billion dollars. And he succeeded in raising money while remaining committed to basic science - research that's aimed at understanding life, not targeted directly at curing diseases.

Despite winning a Nobel Prize and sitting next to Hillary Clinton during a State of the Union address, most people in and out of Washington don't know who he is. The student newspaper at Harvard dubbed him "Dr. Who" after he was selected to give the commencement address, and the customers in his local coffee shop mistook him for a bum when he walked in wearing his old, stinky cycling gear.

Dr. Varmus currently runs the Memorial Sloan-Kettering Cancer Center, where he is President and Chief Executive Officer, in New York City. He is married to Constance Casey, a journalist, and they have two sons, Christopher and Jacob.

Links

Molecular Modeling of ras Protein

Read about the mutations in ras that lead to cancer, and see the protein interact with other molecules in 3D. You'll need the Chemscape Chime 2.0 plugin.

The Bacterial ID Lab

Pick the virtual bacterial ID lab link. This virtual lab takes you to the bench of a sequencing lab where you go through all the steps in sequencing bacterial DNA. Then you search BLAST to find and identify the source of the DNA. (HINT: To copy the sequence during the lab, press 'control' and 'c' simultaneously.)

Molecular Biology and Primate Phylogenetics

This link from the Woodrow Wilson National Fellowship Foundation is a lab protocol for teachers. The lab uses homologous sequences in primates to uncover the evolutionary relationships with our cousins.

Bibliography

Angier, N., 1988, Natural Obsessions: The Search for the Oncogene, Houghton Mifflin Company, pp. 394, Boston.
Barbacid, M., 1987, ras GENES, Ann. Rev. Biochem., 56: 779-827.
Doolittle, W.F., 1999, Phylogenetic Classification and the Universal Tree, Science, 284: 2124-2128.
Foury, F., 1997, Human genetic diseases: a cross-talk between man and yeast, Gene, 195: 1-10.
Kataoka, T., Powers, S., et al., 1985, Functional Homology of Mammalian and Yeast RAS Genes, Cell, 40: 19-26.
Kerem, B., Rommens, J.M., et al., 1989, Identification of the Cystic Fibrosis Gene: Genetic Analysis, Science, 245: 1073-1080.
Ochman, H., Lawrence, J.G., Groisman, E.A., 2000, Lateral gene transfer and the nature of bacterial innovation, Nature, 405: 299-304.
Pandey, A. and Lewitter, F., 1999, Nucleotide sequence databases: a gold mine for biologists, Trends in Biochemical Sciences, July, 276-280.
Powers, S., Kataoka, T., et al., 1984, Genes in S. cerevisiae Encoding Proteins with Domains Homologous to the Mammalian ras Proteins, Cell, 36: 607-612.
Riordan, J.R., Rommens, J.M., et al., 1989, Identification of the Cystic Fibrosis Gene: Cloning and Characterization of Complementary DNA, Science, 245: 1066-1073.
Rommens, J.M., Iannuzzi, M.C., et al., 1989, Identification of the Cystic Fibrosis Gene: Chromosome Walking and Jumping, Science, 245: 1059-1065.
Spector, D.H., Varmus, H.E., Bishop, J.M., 1978, Nucleotide sequences related to the transforming gene of avian sarcoma virus are present in DNA of uninfected vertebrates, Proc. Natl. Acad. Sci., 75: 4102-4106.
Stehelin, D., Varmus, H.E., Bishop, J.M., Vogt, P.K., 1976, DNA related to the transforming gene(s) of avian sarcoma viruses is present in normal avian DNA, Nature, 260: 170-173.
Fallows, James, The Political Scientist, The New Yorker, June 7, 1999.
Anonymous, Biography of Dr. Harold Varmus, http://www.mskcc.org.
autobiography at http://www.nobel.se
autobiography from UCSF website (same autobio at Nobel site)
Malaspina, Rick, New chancellor named for UC San Francisco, February 16, 1998, University of California, Santa Cruz Currents
http://www.ucsc.edu/oncampus/97-98/02-16/ucsf.html.
US Department of Health and Human Services, "Curiosity is the Key to Discovery: The Story of How Nobel Laureates Entered the World of Science," 1992.

Glossary

Mutation - A permanent structural alteration in DNA. In most cases, such DNA changes either have no effect or cause harm, but occasionally a mutation can improve an organism's chance of surviving and passing the beneficial change on to its descendants.
Oncogene - A gene that is capable of causing the transformation of normal cells into cancer cells.

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.
DNA responds to signals from outside the cell.
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.
DNA is only the starting point for understanding human biology.
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