October 21, 2004
New Technique Explores the 'Black Holes' of Chromosomes
A new technique developed by Howard Hughes Medical Institute
researchers significantly reduces the time required to isolate genetic
material from the so-called “black holes” of chromosomes —
the centromeric regions where one chromosome is joined to its sister in
the cell.
Most scientists believe that the centromeric regions of chromosomes
are likely to hold invaluable clues to understanding the machinery that
powers cell division. But those clues have remained largely obscure
because the centromeres contain vast, confusing stretches of repetitive
DNA sequences and segments that shift position in the genome.
“Our approach was the opposite of what a lot of genome projects do, because they try to focus on the genes and exclude the repetitive DNA.”
Daphne Preuss
According to the researchers, the new technique will speed the
process of understanding the function of centromeric regions by
enabling the rapid comparison of these regions of DNA among different
plants and possibly even mammals. Such studies may help researchers
understand the evolution — and thus the function — of the centromeric
regions.
The technique may also enable creation of “designer
chromosomes” in crop plants that could alter their
characteristics to produce desirable traits, according to Howard Hughes
Medical Institute investigator Daphne K. Preuss. She and her colleagues
at the University of Chicago published a research article describing
the technique in the October 2004 issue of the journal Nature
Methods.
Preuss's team reported that they applied the technology to isolate
centromeric DNA from the plant Arabidopsis thaliana, a member of
the mustard family widely used as a model plant in biology.
“The centromeric regions perform a number of essential
functions,” said Preuss. “They include directing
inheritance of a chromosome and keeping the sister chromatids bound to
each other until the later stages of meiosis and mitosis.”
Meiosis is the cell division process that, in the case of plants,
results in the generation of pollen and egg cells; in humans, the
process generates sperm and oocytes. Mitosis is the cell division
process by which organisms increase the number of cells in development
and maintenance. The centromeric region also includes genes important
for preserving the vital function of the region in cell division.
“Despite their importance, the DNA sequencing projects have
often been very challenged in obtaining complete DNA sequence
assemblies in the centromeric region because they are highly
repetitive,” said Preuss. For example, she said, the project to
sequence the Arabidopsis genome took three years of arduous work
by a group of laboratories to develop sequence information for the
plant's centromeric DNA.
To speed that process, Preuss and her colleagues developed an
isolation technique that exploits the fact that centromeric DNA is
distinguished by a high number of methyl groups, which attach to the
cytosine base pairs contained in DNA. Methylation is one method by
which the cell maintains genome regions in an inactive state.
“Our approach was the opposite of what a lot of genome
projects do, because they try to focus on the genes and exclude the
repetitive DNA,” said Preuss. “We wanted to selectively
identify the highly repetitive DNA that corresponds to the
centromere.”
To identify those regions selectively, the researchers used an
enzyme to snip apart the plant's genome, but did not cut the DNA in
methylated regions. By using these resistant fragments to probe the
Arabidopsis genome, the researchers could determine whether they
had identified centromeric DNA. The studies were highly accurate in
identifying centromeric DNA, said Preuss.
“It was a very exciting result,” she said. “We had
spent three years slogging through the centromeres using traditional
methods, which involve sequencing small regions and using them as
probes to `walk' along the DNA to identify others. But with this
method, the results just popped up in three days.”
Once the researchers had identified the centromeric regions in
Arabidopsis, they extended the technique to isolate such regions
in three other plant species that shared a common ancestor with
Arabidopsis. The method succeeded, even though these plants had
different chromosomal numbers and genome sizes than
Arabidopsis.
“We wanted to know if this was a general methodology,”
said Preuss. “Since these other species were evolutionarily
separated from Arabidopsis by different times, it gave us a
beginning in understanding how centromeres evolved.”
The success of the technique with the other species demonstrates
that rapid analysis of the centromeric regions in a broad range of
plants, and even mammals, is feasible. “We believe that this
technique could be extended to mammals, because the one thing that
plants and mammals have in common about their centromeres is the
extensive heavy methylation of centromeric regions,” she said.
“It's certainly worth trying, given that it only takes three
days.”
Analysis of the plant centromeres could have important agricultural
applications, said Preuss. “Having a good understanding of
centromeres means that you can actually make designer
chromosomes,” she said. Such chromosomes could drastically alter
a plant's characteristics to create improved crops plants, said
Preuss.
“In terms of clinical applications, we do know that there are
inherited disorders and cancers associated with centromere function, so
that understanding of how the centromere works could offer important
insights into those disorders,” Preuss added.
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