The serendipitous discovery of a rare mutation and five years of concentrated effort have allowed a team of American and British researchers to define the centromeres of the five chromosomes of Arabidopsis thaliana, a flowering plant that has become the primary model for plant genetics. The centromere is the part of the chromosome that ensures each dividing cell inherits the correct DNA.
The findings, published in the December 24th issue of Science, represent the first time that scientists working with a multi-cellular organism have been able to identify the genetic boundaries of the centromeres -- which are resistant to standard gene mapping techniques -- and to unravel their DNA sequences.
"The centromere is perhaps the most important but also the most inaccessible and the least understood part of a chromosome," said Daphne Preuss, Ph.D., assistant professor of molecular genetics and cell biology at the University of Chicago and leader of the research team.
"Precise mapping of these crucial segments of DNA should shed light on the nature and behavior of chromosomes," said Preuss. "It could help define the centromeres in more complex organisms, boost plant genomics, and speed the development of artificial chromosomes for use in plant engineering," she added. "This now makes Arabidopsis the leading candidate for thorough sequencing of the entire genome in a higher organism."
The Arabidopsis Genome Project hopes to complete sequencing of the first full plant genome by the end of 2000. The sequences of chromosomes 2 and 4 were published in Nature on December 16.
Centromeres are pivotal structures near the center of each chromosome. They are essential for cell division because they form the point of attachment for spindles, the fibers that separate DNA strands into equal piles before cell division.
The DNA near the centromeres is highly condensed, packed and coiled so tightly that it can thwart many types of anaysis. The techniques that allowed genetic and physical mapping of the centromeres in yeast required researchers to analyze the genetic contents of "tetrads" -- clusters of four reproductive cells that are formed from a single parent cell. Unfortunately, tetrads are not stable in most multi-cellular organisms. This has prevented the precise mapping of centromere boundaries.
Other attemps to sequence centromeres have been thwarted by the highly repetitive properties of these regions. Consequently, the Human Genome Project has postponed mapping most of the centromeres until better methods become available.
The crucial breakthrough for studying plant centromeres came in 1994 when Preuss discovered a mutant form of Arabidopsis. The mutation, known as quartet, causes the four products of male meiosis to stick together, forming a tetrad of four pollen grains. This allows scientists, using genetic markers, to monitor how the original five pairs of chromosomes shuffle the deck of genetic information through one replication and two divisions to form the four cells of a tetrad.
The researchers studied more than 1,000 meioses to determine the precise areas at the center of each chromosome in which there was absolutely no exchange of DNA during cell division.
They found, as expected, that the actual centromeres were composed of long arrays of extremely repetitive DNA -- 180 base-pair repeats which have a structural function but contain no genes. This central core, however, was flanked by surprisingly complex DNA composed of diverse sequences.
The researchers had expected to find more of the repetitious, structural DNA bordering the centromere, but they found instead that the surrounding regions contained moderately repetitive DNA, plus multiple "mobile elements" -- DNA that appears to have originate elsewhere and been inserted into the plant's genome -- and several functioning genes.
Being a gene in the centromere region is "like being a satellite in the asteroid belt," suggested Greg Copenhaver, Ph.D., research associate in Preuss's laboratory and first author of the paper. The region is peppered with inserted bits of DNA, from viral or other sources. And because recombination events -- the exchange of DNA between chromosomes -- are limited near the centromere, this part of the chromosome is less able to shed useless DNA than the gene-filled regions farther out on the chromosome arms.
In fact, the researchers found an entire mitochondrial genome embedded near the centromere of chromosome 2. This surprising discovery may shed light on the evolutionary processes that allow DNA from organelles to be exchanged with that from the nucleus.
Understanding the centromeres may prove to be a crucial development for advanced genetic engineering of plants, said Preuss. Previously, researchers could insert only one gene at a time, but by connecting several genes on one long strand to a centromere, it may be possible to move whole sets of genes into an organism. These altered plants, even a weed like Arabidopsis, could then provide an inexpensive source of complex biomolecules for biochemical or pharmaceutical use.
The research was funded by the National Science Foundation, the United States Department of Agriculture, the Consortium for Plant Biotechnology Research, and the David and Lucille Packard Foundation.
The large-scale DNA sequencing of the centromeres was accomplished by the efforts of two teams: one, led by Samir Kaul and Xiaoying Lin at TIGR (The Institute for Genomic Research) was responsible for characterizing the centromere of chromosome 2, and the second, jointly led by Rob Martienssen and Richard McCombie at Cold Spring Harbor and Mike Bevan at the John Innes Institute, Norwich, England, was responsible for sequencing the centromere of chromosome 4. These teams combined their data with the mapping work from Copenhaver and Preuss, defining the boundaries of the centromeres along with their contents.
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