DNA chips in active substance research

In the garden
of molecules

10
Bioinformatics

Active substance
analysts Dr. Edda
Koopmann and
Dr. Peter Eckes
with citrus fruits
susceptible to
fungi.

Crop protection products are be-
coming increasingly effective
and specific. To ensure that this
remains the case in the future,
scientists at Bayer CropScience
searching for suitable sub-
stances distinguish promising
new candidates from those
which work by well-known me-
chanisms, and thus are of little
interest, as early as possible in
their development work. The
scientists rely on an extremely
useful tool to do this: DNA
chips, which provide a
molecular snapshot of a plant
or pest.
U.S. President Bill Clinton hit the head-
lines in February 2001 when he ap-
peared with the molecular biologists
Craig Venter and Francis Collins to
present the first version of the human
genome. The “Book of Human Life”
had been opened, and a new era in
medicine was about to begin.
For scientists working in crop protec-
tion, the event of the century had al-
ready happened two months before. In
March 2000, an international research
team had presented the complete
genome of Drosophila melanogaster,
the most popular model organism
for insects. The publication of the ge-
nome of the thale cress (Arabidopsis
thaliana), an unassuming plant which
is nonetheless a key botanical model
organism of great scientific interest,
followed in December. Here again, the
complete sequencing of the hereditary
Active genes:
a test substance
is sprayed onto
thale cress.
blueprint of a living organism opened
up unimagined possibilities. Thanks to
the growing body of genetic informa-
tion, which now includes the complete
or almost complete genomes of other
plants, some insects and fungi, sophis-
ticated biotechnical methods now al-
low scientists to deliberately search for
further principles of action in the fight
against pests of every kind.
This is because if a new substance
works according to a previously un-
known biochemical principle, it is very
likely to be effective in controlling
fungi, insects and weeds that have be-
come resistant to traditional products.
Furthermore, understanding molecular
processes in detail also allows scien-
tists to assess very accurately whether
a potential substance will meet the
strict environmental and safety re-
quirements imposed on modern crop
protection products.
Bayer’s scientists were quick to pick
up on the new opportunities. “Once
the Arabidopsis genome had been de-
coded, we thought that now it might
make sense to conduct detailed inves-
tigations into changes in genetic ac-
tivity triggered by the action of crop
protection products,” remembers Dr.
Peter Eckes, a bioinformatician work-
ing in Bayer’s herbicide research facili-
ties in Frankfurt am Main. He and his
colleagues in Bayer’s crop protection
research therefore started to plan and
conduct the first experiments with DNA
chips representing Arabidopsis genes.
These diagnostic marvels, although
scarcely bigger than a thumbnail, reg-
Applications for DNA chips
To investigate principles of action
This is the most important task. It involves
comparing the reaction of model organisms to
active substance candidates with their reac-
tion to substances with known principles of
action. This gives an early indication of how
the substance candidate works. If the reaction
does not match any known pattern, bio-
informaticians use the gene activity profile
to search for clues to the metabolic paths in-
volved. This makes it much easier to under- 11
stand the biochemical principle of action than
Bayer research 17
would otherwise be the case.
To investigate resistance
Comparing the gene chip pattern of a resist-
ant organism to that of a normal organism
provides vital information as to the causes of
resistance.
To search for pro-drugs
As DNA chips react in the same way as entire,
healthy pest organisms, they are also sensitive
to pro-drugs, substances which remain inac-
tive until they reach the desired site of action
where they transform into an active form,
usually because an enzyme in the organism
changes them.
To conduct in vitro / in vitro comparisons
Sometimes trial substances are outstandingly
effective on the isolated target enzyme in bio-
chemical tests in vitro, but fail when confront-
ed with the entire organism. Comparing the
gene activity profile of the ineffective candi-
date with that of an effective substance can
help clarify why this is the case.
To conduct gene function analysis
Many parts of the genome have no function,
and only a few genes are essential to life.
DNA chips help us find these vital genes and
so focus on potential sites of action.
To investigate toxicology
DNA chips can often be used to ascertain at a
very early stage whether a potential substance
is likely to cause unwanted side effects.

Section of a DNA chip
12
ister a snapshot of all the genes that
Bioinformatics
are switched on, and therefore active
in the production of proteins, within a
cell at a given time.
Chip analysis supports research
into resistance
Once the biologists know which genes
are active, they can investigate what
nutrients, enzymes or receptors the cell
Dipl. Ing. Catherine urgently needs at that time. These are
Sirven uses com- highly significant details: for example,
plicated statistical
methods for gene if genes for several enzymes involved
analysis. in a particular metabolic path are ac-
tivated following the application of a
crop protection product, the obvious
conclusion is that the herbicide un-
Computer evaluation
der investigation probably blocks that
path. The fact that the plant is trying
to counter this by upregulation of the
metabolic pathway genes is an impor-
tant key to the substance’s mode of
action. But that’s not all: “Gene chips
are a very promising option in resolv-
ing a number of problems, for instance
when we look into the causes of re-
sistance,” says Dr. Edda Koopmann,
head of the bioinformatics laboratory
at Bayer CropScience in Monheim. A
fungus that is resistant to a particular
fungicide often activates genes for pro-
teins that break down the substance
into harmless components or remove
it from the cell. In insects that toler-
ate a crop protection agent, a specially
developed DNA chip could be used as
a diagnostic tool to help elucidate this
resistance.
This is why the bioinformaticians who,
along with molecular biologists, devise
and assess the DNA chip experiments,
see themselves as providing a serv-
ice to the CropScience biologists and
chemists in their attempts to solve
tricky problems. “We evaluate the chip
experiments, a statistically demanding
task, provide additional information
and consult closely with the research
scientists,” says Dr. Koopmann. That is
why bioinformaticians are on hand at
all Bayer CropScience research sites:
in Monheim, where work is concen-
trated on insecticides but also certain
aspects of fungicides, in Frankfurt am
Main, the center of herbicidal activity,
and in Lyons, which is the main site for
fungicidal research.
RNA molecule from the organism
with luminescent material (yellow)
Gene fragment captor
RNA molecule binds specifically
Gene in the organism is active
Novel substances can be identi-
fied at an early stage
“Good contacts with the biologists are
the most important thing,” explains
Catherine Sirven, who works in Lyons.
The onus is very much on the compu-
ter experts’ communication skills: they
have to clearly understand the prob-
lems facing the researchers if they are
to find the right solutions. Adds Sirven,
“In Lyons we carry out research on
fungi. These organisms are particularly
difficult to investigate because they are
so varied, and each one goes through
different stages in its lifetime.” She and
her colleagues discuss the findings for
new potential fungicides whose princi-
ples of action are unknown: “Genetic
chips of the rice blast fungus and ad-
ditional biochemical experiments al-
low us to see what changes the new
substance causes in the fungi and to
compare the results with the response
of these fungi to existing substances.”
If the team finds out that the candidate
works in much the same way as an ex-
isting substance, then further research
would be pointless. If not, things can
get interesting: “If the mode of action
is one that was previously unknown,
the substance could be successful,”
explains Sirven.
A single chip contains up to 500,000
snippets of genes which wait like
fish-hooks for pieces of genes which
match them. To give an example, the
gene snippets on an Arabidopsis chip
are made up of defined pieces of
the plant’s genome, which comprises
How a gene chip works
A DNA chip contains up to 500,000
gene fragments arranged in grids.
These capture RNA molecules formed
in a cell by active genes. After an
experiment in which an organism is
treated with a crop protection product,
the RNA is isolated from the organism,
prepared and treated with luminescent
material. This marked RNA is dripped
onto the DNA chip and each RNA
molecule binds to the place on the
chip where its gene fragment captor
is located. An assessment of the chip’s
light pattern then determines which
genes in the organism were active. The
chip has multiple binding sites for each
gene in order to prevent errors and
assess the intensity of gene activity.
around 24,000 genes. The scientists
then extract the ‘messenger RNA’ mol-
ecules from a plant cell; the type and
number of these molecules reflect the
level of gene activity. These are also
fragmented and applied to the DNA
chip after being treated with lumines-
cent material.
A complete overview of what
happens in the cell
The scientists note which fish-hooks
light up because they have caught a
piece of RNA. This shows them which
genes in their probe were active. One
single chip is not that helpful, however.
“What really makes the experiment
exciting is the comparison,” says Dr.
Koopmann. “Gene chips are critical
if we want to investigate differences
between two situations.” In another
experiment she compares probes from
several fungi cultures treated with test
substances. These substances have
proven to be effective in biochemical
tests, but are inactive in the fungal
culture for unknown reasons. “These
comparisons show us how well in vitro
experiments transfer to natural, in vitro
situations,” she explains.
Real bioinformatics work is therefore
not actually required with DNA chips
until the results are available in the
form of a gene activity pattern. Reduc-
ing vast quantities of data, so complex
as to be almost chaotic, to the key in-
formation is a task requiring complex
statistical evaluation, fast computers
and higher mathematics. As Dr. Marcus
13
Weidler, who together with Dr. Peter whose mode of action is still not under- Chip evaluation:
Bayer research 17
Active genes
Eckes laid the foundations for today’s stood. Our task over the next few years become glowing
statistical evaluation of the chip experi- will be to analyze them.” dots in the com-
ments, is well aware, “The experiments puter analysis
(background).
contain so much information that you www.affymetrix.com
could easily be swamped by it.” But The website of the world’s larg-
this does have advantages: “We opted est DNA chip manufacturer offers a
for gene chips because, firstly, they give wealth of information on all aspects
us the opportunity to have an overview of this new technology.
of changes in the organism and, sec-
ondly, as with a high-resolution satel-
lite image, they allow us to get down
to the fine detail at the right points
quickly.”
Considerable experience and standard-
ized operating processes are needed to
work with gene chips, which are highly
sensitive as a result of their complex-
ity. “After a while you can even tell
which colleague carried out the tests,”
says Dr. Koopmann. “It’s a task that
DNA chip reveals mode of action of a new substance
requires great sensitivity and good,
self-checking software to interpret the A test substance was sprayed onto four plants. In-
data correctly.” The bioinformaticians creased activity of the six genes shown here relative
to the four control plants was then determined.
in Frankfurt, Lyons and Monheim are As these genes are related to the plant growth
old hands at this by now: they select substance auxin, it can be concluded that the test
different types of chips from different substance imitates the effect of auxin.
manufacturers according to the task at
hand and have developed their own Gene activity Gene activity
Bayer-specific work processes for a 104 104
whole range of different issues. 103 103
And they have been successful. For
102 102
example, they have built up a compre-
hensive compendium for insecticides 10 10
and herbicides, used to rapidly allo- 1 1
cate each new potential substance to 0.1 0.1
a specific principle of action - or not, 1 2 3 4 1 2 3 4
as the case may be. “And that’s when
Four experiments with- Four experiments with
things really take off,” says Dr. Eckes. out the test substance the test substance
“We have a whole range of substances