Concept

The DNA sequences from two individuals of the same species are highly similar — differing by only about one nucleotide in 1,000. Each DNA difference results from a mutation — ranging from single nucleotide changes, to small repeated units, to larger insertions and deletions. Some mutations generate novel changes that are starting points of evolution, and some are responsible for disease. In humans, the vast majority of mutations occur in DNA regions that do not encode proteins. Most of these are neutral in terms of evolution or health; they have no negative or positive effect. In the 1920s, DNA mutations were first induced in Drosophila using X-rays. Other types of ionizing radiation were also found to produce mutations. Ultraviolet radiation, a component of sunlight, causes specific kinds of DNA damage, including the linking of adjacent thymine nucleotides. Chemicals from a variety of man-made and natural sources are known mutagens. Also, DNA replication, itself, is not perfect and is a source of new mutations.

Animation

I’m Roy Britten. In the 60s, David Kohne and I found that mouse cells contain multiple copies of very similar DNA sequences. We did this by looking at the reassociation rates of DNA strands. Let me explain. We already knew that DNA encodes proteins. Genetic information is stored in the sequence of nucleotides of a DNA strand. The nucleotides pair through hydrogen bonds — A with T, and G with C — to form two complementary strands. This is the DNA double helix. DNA can be extracted from prokaryotes and sheared into smaller fragments. When heated to near boiling, the hydrogen bonds between the complementary base pairs are disrupted, and the double stranded DNA dissociates into single strands. When the temperature drops about 25º below the dissociation temperature, the DNA strands reassociate. Random collisions bring complementary strands back together, and the hydrogen bonds reform. Perfect matches don't always occur. The faster the temperature is lowered, the less time there is for complementary strands to "find" each other. There can be local areas of complementarity. We measured the DNA reassociation time by taking advantage of differences between double and single stranded DNA. Double stranded DNA has a higher affinity for a crystalline form of calcium phosphate called hydroxyapatite. A column filled with hydroxyapatite traps double stranded DNA but allows single stranded DNA to flow through. Conditions can be set so that the column retains double stranded DNA with some mismatches. All the DNA can be washed off the column, and both single and double stranded DNA amounts can be measured. In 1962, reassociation reactions were done using eukaryotic DNA. Compared to bacteria like E. coli, eukaryotes like mice have about 100 times more DNA in their cells. Therefore, we were surprised that some eukaryotic DNA reassociated faster than E. coli DNA. We decided to analyze the data a bit further. We plotted the fraction of reassociated DNA against the log of the product of DNA concentration and time (C0t). Using the same inital concentration of DNA, C0, we compared the reassociation rates between organisms with different genome sizes. For polyU and polyA strands, the C0t curve looked like this: The C0t graph for E. coli DNA looked like this. Notice that the curve was displaced to the right. The reassociation reaction takes longer to complete because the E. coli DNA is far more complex. Unlike polyU and polyA strands, the DNA strands of E. coli take longer to find the right match. The reassociation curve of a portion of mouse DNA called satellite DNA looked like this. Notice how the mouse DNA reassociated faster than E. coli DNA. It turns out that mouse satellite DNA contains lots of repeated sequences. These sequences are so similar that they reassociate easily; there are no unique sequences that need to hunt for their partners. We found that an average eukaryotic genome had a reassociation curve that looked like this: The first part of the curve is the fast component and represents highly repetitive DNA that reassociates very quickly. Highly repetitive DNA can make up about 25% of the genome. The second part of the curve is the intermediate component where the middle/moderately repetitive DNA reassociates. This can represent about 30% of the DNA in the eukaryotic genome. The third part of the curve is the slow component; there is no repetitive DNA in this fraction. The slow component can make up to 45% of the DNA in the genome. We tested these DNA types to see which fraction coded for protein. We added radioactive mRNA as a tracer to the beginning of a reassociation reaction. As the temperature dropped, the mRNA hybridized with its template DNA. No mRNA hybridized to the highly repetitive DNA fraction. Very little of the radioactive mRNA hybridized with the mid-repetitive DNA fractions. Most of the radioactive mRNA hybridized with the slow DNA fraction — giving us a rough approximation of the fraction of a genome that encodes protein. The green curve represents the hybridization of the radioactive mRNA. If repetitive DNA doesn’t code for proteins, where did it come from and why is it there? Repetitive DNA probably arises from errors in DNA replication. The most highly repetitive DNA is usually found in regions near the centromeres and may have a function in chromatid pairing during cell division. Highly repetitive DNA is composed primarily of very short "tandem repeats" — numerous repeated units lined up head-to-tail, like the cars of a train. The repeated unit may be as short as two nucleotides ... ... to about 20 nucleotides. Moderately repetitive DNA is composed of larger elements scattered widely throughout the genome. Two major groups are categorized by size: Short Interspersed Elements (SINEs) are several hundred nucleotides in length, while Long Interspersed Elements (LINEs) are several thousand nucleotides long. Both groups are derived from transposons, so-called "jumping genes," which have accumulated over evolutionary time by moving to new chromosome locations. LINE elements jump using reverse transcriptase, which functions in the same way but is not closely related to retrovirus RT. SINEs are elements that do not produce their own reverse transcriptase. They "borrow" reverse transcriptase from LINEs or other sources. Even though they use the same enzyme for insertion, LINEs and SINEs favor different insertions sites. Humans and other primates have about 500,000 copies of Alu, a 300-bp SINE. Alu elements alone are believed to make up about 5% of the human genome — an amount equal to coding sequence!

Gallery

Roy Britten, 1970s.

Roy Britten with his two sons and his aunt, 1970s.

Audio/Video

Video Interviews

Roy J. Britten

Roy Britten is a Distinguished Carnegie Senior Research Associate, Emeritus, at the California Institute of Technology.

Clip 1 (1:09)
Using mouse satellite DNA to study reassociation kinetics.

Clip 2 (0:29)
Biologists and the stress of math.

Clip 3 (0:29)
Composition of repeated DNA in the genome.

Clip 4 (0:46)
Are repeated DNA species specific?

Biography

Roy Britten did seminal research on repetitive DNA and its evolutionary origins.

ROY JOHN BRITTEN (1919-)

Roy Britten was born in Washington D.C. His mother worked at the National Research Council and his father was a statistician. Britten was exposed to science early on. Growing up, Britten and his brother shared a basement chemistry lab. He also frequented the public exhibits in the rotunda of one of the National Academy buildings, where he could see the working of a Foucault pendulum and learn about sunspots.

In 1940, he went to the University of Virginia to study physics. Not long after, he was recruited to work on the Manhattan Project. He didn't return to school until 1946. He went to Princeton to do graduate work in nuclear physics.

By the time he finished his Ph.D. in 1951, Britten had decided that the world of nuclear physics had changed. He made plans to do post-doctoral work in biophysics at the Department of Terrestrial Magnetism in the Carnegie Institution in Washington. He took the phage course at Cold Spring Harbor Laboratory to brush up on his biology, and started working on the kinetics of DNA hybridization with the group at the Carnegie. Through this work, Britten showed that eukaryotic genomes have many repetitive, non-coding DNA sequences.

Since his work on repetitive DNA, Britten has been interested in evolutionary biology, specifically the nature of repetitive DNA and its origin and evolutionary history. He has done work on human repetitive DNA elements like Alu, and repetitive DNA elements in sea urchins - a candidate organism for the sequencing project. He is also looking at other repetitive elements in the human genome from data generated by the Human Genome Sequencing Project.

Britten has been at the California Institute of Technology since 1970. He is part of the gene regulation research group and is a Distinguished Carnegie Senior Research Associate, Emeritus. He is also an adjunct professor at the University of California, Irvine.

Britten has a number of hobbies and interests outside of science. He has been a long-time sailor and musician; he plays the flute though admits that lately he hasn't had the time. Britten paints "oils, because water-color is too difficult." And to keep up with the times, he has been generating computer art. He writes science fiction. His artwork and fiction are currently still private.

Links

Genetic Origins

Try our online site with laboratories and exercises that use Alu -- a type of repetitive DNA -- to track human populations and migrations.

Bibliography

Alberts, Bruce et al., 1983, Molecular Biology of the Cell, Garland Publishing Inc., New York.
Britten, R.J., and Kohne, D.E., 1968, Repeated Sequences of DNA, Science, 161: 526-640.
Britten, R.J., and Kohne, D.E., 1970, Repeated Segments of DNA, Scientific American, 222: 24-31.
Darnell, J., Lodish, H., and Baltimore, D., 1986, Molecular Cell Biology, Scientific American Books, Inc., U.S.A.
Raven, P.H., and Johnson, G.B., 1986, Biology, Times Mirror/Mosby College Publishing, St. Louis.
Lewin, Benjamin, (1990), Genes IV, Oxford University Press, New York.

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 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.
Living things share common genes.
DNA is only the starting point for understanding human biology.
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