Concept


In most cases when DNA is extracted from living cells, the proteins (including histones) are dissolved away. This results in long strands of naked DNA, which retain their genetic information. So it is useful to visualize a chromosome as a continuous strand of DNA. Arrayed along the DNA strand are the genes, specific regions whose sequences carry the genetic code for making specific proteins. The genes of bacteria are tightly packed together; virtually all the DNA encodes proteins. However, experiments done in the 1960s, showed that a large proportion of eukaryotic DNA is composed of repeated sequences that do not encode proteins. Long non-coding sequences — or intergenic regions — separate relatively infrequent "islands" of genes. Research in the 1970s, showed that numerous non-coding sequences — introns — are also found within genes, interrupting the protein-coding regions, or exons. It is estimated that only about five percent of human DNA encodes protein.

Animation


Hi, I’m Roger Kornberg. Aaron Klug and I were interested in a class of proteins called histones and how they interact with DNA. There are five different kinds of histones. Histones bind to DNA to form the chromatin ("colored material") in the nucleus of higher cells. In non-dividing cells, the chromatin is dispersed throughout the nucleus. During prophase of cell division, the chromatin condenses into the visible structures we know as chromosomes. This electron micrograph shows a cell in metaphase; the chromosomes are lined up in the middle of the cell. The presence of histone proteins in the nucleus of higher cells was part of a debate in the 1940s about which molecule, DNA or proteins, is the hereditary material. Of course, DNA turned out to have that distinction. However, X-ray diffraction studies later showed that histones play an important role in providing structure for the DNA helix. Remember Maurice Wilkins? In 1964, he and Vittorio Luzzati noticed that chromatin has a repeating pattern with intervals of about 100 angstroms (1Å = 10-10 m). This repeat is different from the repeating patterns of DNA itself. Aaron Klug also saw similar X-ray diffraction patterns in chromatin. This repeat suggested that histones play a role in "packaging" DNA. I'm Dean Hewish. I'm Leigh Burgoyne. In 1973, while we were at Flinders University in South Australia, we got a result that supported the idea of DNA packaging. We isolated the enzyme DNA nuclease from rat liver cells and used it to digest chromatin. We electrophoresed the digested chromatin material ... ...and we saw a regular pattern of bands on the gel. This is a photo of the gel we ran in 1973. We figured out that the bands were multiples of the smallest size fragment, later determined to be about 200 base pairs (bp). So these repeated bands corresponded to 200 bp, 400 bp, 600 bp, 800 bp, and so on. We concluded that the histones are distributed evenly on the DNA and, at the points where they bind, protect the DNA from nuclease digestion. This is completely different from the digestion pattern of "naked" DNA without histones. Naked DNA digested with nuclease produces a smear of thousands of different-sized fragments. Based on the X-ray diffraction patterns and the nuclease experiments, chromatin was proposed to be DNA and the histone cores it wrapped around. The 200 bp repeat observed after nuclease digestion corresponds to 200 bp of DNA wrapped around each histone core. The 100 Å measurement from X-ray diffraction patterns is the width of the histone core and the DNA. My colleagues and I did experiments that confirmed this model, and we also figured out the arrangement of the histones in the core. We individually purified the histones from the DNA. We found that H2A and H2B tend to stick together, as do H3 and H4. If we mixed the H2A/H2B complex with the H3/H4 complex, and then add naked DNA, we got the same X-ray pattern as for chromatin. More analysis revealed that each histone core has 8 proteins — two copies each of the H2A/H2B and H3/H4 complexes. This histone core with wrapped DNA is called a nucleosome. This is an electron micrograph of chromatin. The "string" is called the 10 nm fiber. The "beads" are the nucleosomes. But where is the H1 histone? It turns out H1 is not part of the histone core. Instead, it binds between nucleosomes to give even more structure to chromatin. H1 is sits just outside of each nucleosome and interacts with the H1 in the next nucleosome. At higher salt concentrations, the 10 nm fiber is further compacted into the 30 nm fiber. The DNA helix is already twisted. By adding twists to make these nucleosomes and solenoid structures, the DNA is supercoiled. Even more organization is involved in maintaining the condensed chromosome. Loops of DNA are attached to a protein scaffold made up of several non-histone proteins. This scaffold maintains the shape of a chromosome — even in the absence of histones. Chromosomes are really one continuous piece of DNA. In this electron micrograph you can see the DNA strand from one chromosome after the histones have been removed. Up to 6 feet of DNA is packaged to fit into the nucleus of one cell. The DNA is first wrapped around histone cores to form nucleosomes and the 10 nm fiber. The 10 nm fiber is further coiled into the 30 nm fiber, where six nucleosomes make one turn. The 30 nm fiber is then looped onto protein scaffolds when chromosomes condense.

Gallery


The Kornberg family, Stockholm, 1959. (L-R) Roger, Kenneth, Sylvy, Arthur, Thomas.
Roger Kornberg in his laboratory, 1970s.
Aaron Klug at a Cold Spring Harbor meeting.
Roger Kornberg, 1980s.
Aaron Klug at a Cold Spring Harbor meeting.
Dean Hewish, 1973.
Leigh Burgoyne, 1973.
Photo of chromatin digested by nuclease, from Hewish and Burgoyne's 1973 experiment.
Electron micrograph of the 10-nm fiber.
Electron micrograph of the 30-nm fiber.

Audio/Video


Audio Glossary

Chromosome

Video Interviews

Roger Kornberg

Dr. Kornberg is a Professor of Structural Biology at Stanford University's school of medicine. He did pioneer work on chromatin structure, and is now working on gene regulation and control.

Clip 1 (0:37)
Early experiences with science.

Clip 2 (0:51)
Hierarchy of levels of chromatin condensation.

Clip 3 (1:15)
Packing ratio of DNA.

Clip 4 (0:32)
Mechanism of condensation -- accordion vs. ball of yarn.

Clip 5 (0:51)
Mechanism of regulation for prokaryotes vs. eukaryotes.

Biography


 

In 1974, Roger Kornberg worked out the importance of histones to chromatin structure.

ROGER KORNBERG (1947-)

Roger Kornberg was born in St. Louis, Missouri. He was the first of three children born to Arthur Kornberg and his wife, Sylvy. With both parents being well-respected scientists, it was not surprising that Roger Kornberg also developed an interest and an enthusiasm for science. As he said in an interview:

Science was a part of dinner conversation and an activity in the afternoons and on weekends. Both my parents had fine scientific minds and taught by example how to approach questions and problems in a logical, dispassionate way. Scientific reasoning became second nature. Above all, the joy of science became evident to my brothers and me.

Kornberg studied chemistry and biochemistry, and without having to think about it, became a scientist. He graduated from Harvard University in 1967 with a Bachelors in science, and went to Stanford University for graduate work. His doctoral thesis was on the chemical nature of phospholipids.

In 1972, Kornberg went to the Medical Research Council in Cambridge for postdoctoral work in X-ray crystallography. There he became interested in the X-ray patterns Aaron Klug obtained for chromatin. Using this and other experimental data, Kornberg eventually worked out the importance of histones to chromatin structure. Kornberg published his results in 1974.

Kornberg stayed on staff at the MRC until 1975 when he was offered an assistant professorship at Harvard University. In 1978, he moved to Stanford University where he is now professor of structural biology.

Over the past 35 years, Kornberg has published over 150 peer-reviewed research papers on phospholipid and chromatin structure, gene regulation and transcription control. His current research interest is on the overall structure of chromatin and chromosomes and how such structuring may be associated with gene repression.

Kornberg used to play the violin as a child and likes to read. However, he admits that his main interest outside of science revolves around his family.

Factoid

Links


 

Links

The Making of a Chromosome

This site has nice renderings of chromatin and nucleosomes.

Modelling Chromatin

From Lutz Ehrlich's group at EMBL, this site has MPEG animations on chromatin modelling.

A Microscopist View of Chromosome Organization

From Gwen Childs at the University of Texas Medical Branch, this web site explains chromosome organization using electron micrographs. Dr. Childs is the program director of the cell biology graduate program, and put together this extensive site on cell biology for students.

Bibliography

  • Alberts, Bruce et al., 1983, Molecular Biology of the Cell, Garland Publishing Inc., New York.

  • Darnell, J., Lodish, H., and Baltimore, D., 1986, Molecular Cell Biology, Scientific American Books, Inc., New York.

  • Finch, J.T., Lutter, L.C., Rhodes, D., Brown, R.S., Rushton, B., Levitt, M., and Klug, A., 1977, Structure of Nucleosome Core Particles of Chromatin, Nature, 269: 29-36.

  • Kornberg, Arthur, 1989, For the Love of Enzymes, Harvard University Press, Cambridge.

  • Kornberg, R.D., 1974, Chromatin Structure: A Repeating Unit of Histones and DNA, Science, 184: 868-871.

  • Kornberg, R.D., and Klug, A., 1981, The Nucleosome, Scientific American, 244: 52-64.

  • Kornberg, R.D., and Lorch, Y., 1999, Twenty-five Years of the Nucleosome, Fundamental Particle of the Eukaryote Chromosomes, Cell, 98: 285-294.

  • Lewin, Benjamin, 1990, Genes IV, Oxford University Press, New York.

  • Raven, P.H., Johnson, G.B., 1986, Biology, Times Mirror/Mosby College Publishing, St. Louis.

Glossary


Chromosome - One of the threadlike "packages" of genes and other DNA in the nucleus of a cell. Different kinds of organisms have different numbers of chromosomes. Humans have 23 pairs of chromosomes, 46 in all: 44 autosomes and two sex chromosomes. Each parent contributes one chromosome to each pair, so children get half of their chromosomes from their mothers and half from their fathers.

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