The discovery in yeast cells of a
genetic network that guards
against lethal DNA damage is a
first step in the creation of a database of
disease-causing combinations of mutated
human genes, according to School
of Medicine researchers led by Jef D.
Boeke.
In a report in the March 10 issue of
Cell, the Johns Hopkins team described
a genetic network that is necessary for
ensuring genomic stability in yeast. This
study also identified previously unrecognized
genes critical for maintaining
DNA integrity and novel functions for
well-known genes.
"A lot of human diseases are caused
by multiple gene mutations that are difficult
to identify," said Boeke, who is a
professor of molecular biology and genetics
and director of the High Throughput
Biology Center at the School of Medicine.
The yeast cell is an excellent model
for this kind of study, Boeke said,
because 25 percent of human disease
genes are also found in yeast. Therefore,
the discovery of this network of genes
could help to identify mutations whose
combined deleterious effects cause human
diseases, including cancer and neurodegeneration,
as well as aging.
"The interactions we discovered in
yeast could also help researchers select the
human versions of these genes suitable as
targets for the development of new, more
targeted and less toxic cancer therapies,"
Boeke said.
The goal of the Johns Hopkins study was
to identify pairs of genes that, while different,
play redundant roles in governing
genomic integrity in yeast cells, filling in for
each other when one of the genes is mutated
or deleted. Such redundancies ensure that
each task in the network of biochemical
reactions governing DNA stability is accomplished,
Boeke noted.
Based on the data from this study, the
investigators were able to separate the genes
governing the stability of yeast DNA into 16
modules, or mini pathways of genes, based
on these genetic interactions, which are
called synthetic fitness or lethality interactions.
Synthetic lethality is a phenomenon in
which two mutations that are not individually
lethal cause cell death when combined.
Specifically, the Johns Hopkins
team identified 4,956 interactions among
875 genes involved in DNA repair, DNA
replication, the halting of replication and
cell cycle progression by "checkpoints" so
that damaged DNA can undergo repair,
and responses to oxidative stress necessary
for reducing the intracellular levels of
highly reactive molecules that bind to and
damage DNA.
The yeast has about 6,000 genes, of which
'Genetic network' in yeast guards against lethal DNA damage
about 1,000 are essential to survival and
5,000 are not, Boeke said. Specifically, 1,000
of the 5,000 nonessential genes are important
enough so that the yeast grows slowly
if any one of them is absent. And any of
the 4,000 other genes can be deleted from
the cell without interfering with the cell's
growth.
A major goal of the Johns Hopkins team
is to determine which of the non-essential
genes interact, Boeke said. All such
pairwise combinations of the 5,000 nonessential
genes in the yeast genome would
require about 25 million tests, he said. In
the current study, 74 genes were tested
in pairwise combination with the 5,000
nonessential genes, a feat approximately
equivalent to 370,000 gene pair tests.
The Johns Hopkins team used a technology
known as dSLAM (heterozygote
diploid-based synthetic lethality analyzed by
microarray) to look at the effects of 5,000
different double mutations on cell fitness
in a single experiment. With this technology,
only 5,000 tests would be required to
map the 25 million pairwise combinations,
greatly speeding the work.
The dSLAM strategy is somewhat like
pulling out parts of a radio at random to see
what happens, Boeke said.
"With yeast, as with a radio, you might
rip out part A or part B and find that the
radio still works; but if you pull out both
parts and the radio dies, you would learn
that A and B can compensate for each
other's absence. The parts we're pulling out
of yeast are genes, and we look to see what
happens when both of the genes are pulled
out."
The dSLAM technology takes advantage
of DNA barcode that identifies
which genes a yeast cell is missing. This
is much like using a commercial barcode
in a store to quickly identify items at the
checkout counter.
The scanner in this case is a microarray:
a grid of thousands of spots on a
piece of glass that holds a unique "sensor"
strand of DNA that matches one of
the barcodes. Machines then read the
microarray to identify which of the sensors
found matching barcodes that identified
specific yeast cells with specific
mutations. If two genes that compensated
for each other are knocked out,
the yeast cell dies and the microarray
doesn't record that cell, Boeke noted.
That means the two genes interact, he
said.
"This strategy for finding interacting
genes will open the door to an extraordinarily
rich source of new data on DNA
damage, repair and human diseases,"
Boeke added.
This work was supported by the
National Human Genome Research
Institute, a National Institutes of Health
Roadmap grant and the Whitaker Foundation.
Other contributors to this paper
include Joel S. Bader, an assistant professor;
Xuewen Pan, the first author of
the paper and a postdoctoral fellow;
Ping Ye, a postdoctoral fellow; and Daniel
S. Yuan, a research associate. All
work in the new interdisciplinary High
Throughput Biology Center at Johns
Hopkins.