Robert J. Brooker - Genetica
Esperimento di genetica 21.1
The Coordinate Regulation of Many
Genes Is Revealed by a DNA Microarray
Analysis
One way that cells respond to environmental changes is via the coordinate regulation of genes. Under one set of environmental conditions, a particular set of genes may be induced, while under another
set of conditions, those same genes may be repressed. In the past,
researchers have been able to study this type of gene regulation using tools that can analyze the expression of a few genes at a time.
The advent of microarrays, however, has made it possible to study
the expression of the whole genome under different sets of environmental conditions.
One of the first studies using this approach involved the analysis
of the yeast genome. The genome of baker’s yeast, Saccharomyces
cerevisiae, was the first eukaryotic genome to be sequenced. It encodes approximately 6,300 different genes. An important process in
the growth of yeast cells, as well as cells of other species, is the
ability to metabolize carbon sources using different metabolic
pathways. When yeast cells have glucose available, they metabolize
the glucose to smaller products in a process called glycolysis. If
oxygen is present, these products can be broken down via the tricarboxylic acid cycle (TCA or citric acid cycle) that occurs in the
mitochondria. Therefore, when yeast are first given glucose and
then allowed to metabolize it in the presence of oxygen, they first
metabolize the carbohydrate via glycolysis, and then, when the glucose is used up, they metabolize the products of glycolysis via the
TCA cycle. The process of switching from glycolysis to the TCA
cycle, called a diauxic shift, involves major changes in the expression of genes involved with carbohydrate metabolism. The goal of
the experiment described in Figure EG21.1.1 was to identify genes
that are induced and repressed as yeast cells shift from glycolysis to
the TCA cycle. It was carried out by Joseph DeRisi, Vishwanath
Iyer, and Patrick Brown in 1997.
As shown in Figure EG21.1.1, yeast cells were initially given
glucose as their carbon source for growth and then allowed to grow
for several hours. Over time, the glucose was used up, and the cells
shifted from glycolysis to the TCA cycle. At various time points,
samples of cells were removed, and the RNA was isolated. The
RNA was then exposed to reverse transcriptase, poly-dT primers,
and deoxyribonucleotides, one of which was fluorescently labeled.
This created fluorescently labeled cDNAs.
To determine the relative changes in RNA synthesis, two different fluorescent dyes were used. The RNA collected at the first time
point—when glucose was at a high level—was used to make cDNA
that contained a green fluorescent dye. The RNA collected at later
time points was used to make cDNAs that contained a red fluorescent dye. A sample of green cDNA (from the first time point) was
then mixed with a sample of red cDNA (from later time points), and
the mixture was hybridized to a microarray containing about 6,200
yeast genes. A laser scanner was used to measure the amount of red
fluorescence and green fluorescence at each spot in the microarray.
The fluorescence ratio (red fluorescent units divided by green fluorescent units) provided a way to quantitatively determine how the
expression of the genes was changing. For example, if the ratio was
high, this means that a gene was being induced as glucose levels
fall, because the amount of red cDNA would be higher than the
amount of green cDNA. Alternatively, if the red to green ratio was
low, this means that a gene was being repressed as glucose is used
up.
THE GOAL
A diauxic switch from glycolysis to the TCA cycle will involve the
induction of certain genes and the repression of other genes. The
goal was to identify such groups of genes.
ACHI EV I N G TH E GOAL — FI GU RE EG2 1 .1.1 T he use of a DN A mic roa rra y t o st udy c a rbohydra t e m e t a bolism in ye a st.
Starting material: A commonly used strain of Saccharomyces cerevisiae (baker’s yeast) was used in this study. The researchers had made
a DNA microarray containing (nearly) all of the known yeast genes.
© 2010 The McGraw-Hill Companies, S.r.l. - Publishing Group Italia
Robert J. Brooker - Genetica
© 2010 The McGraw-Hill Companies, S.r.l. - Publishing Group Italia
Robert J. Brooker - Genetica
THE DATA*
* Green spots are genes expressed early in growth, while red
spots are expressed later. Yellow spots are expressed more
evenly. Spots that are barely visible indicate genes that are not
substantially expressed under these growth conditions.
© 2010 The McGraw-Hill Companies, S.r.l. - Publishing Group Italia
Robert J. Brooker - Genetica
INTERPRETING THE DATA
A portion of one microarray is shown at the beginning of the data of
Figure EG21.1.1. As seen here, the array shows many spots, some
of which are relatively green and some of which are relatively red.
The green spots indicate genes that are expressed at higher levels
during early stages of growth when glucose levels are high. An example is a gene designated TEF4, which is involved with protein
synthesis. Red spots identify genes that are expressed when the glucose is depleted. The gene GLK1 encodes an enzyme that phosphorylates glucose but is expressed only when glucose levels are
low. In addition, many spots are not fluorescent, indicating there
was not much cDNA in the sample to hybridize to the DNA strands
in those locations. These spots would correspond to genes that are
not greatly expressed under either condition. Judging from the
numbers of red and green spots, the shift from glycolysis to the
TCA cycle involved a great amount of gene induction and repression. By determining the red:green ratio at each spot, it was found
that 710 genes were induced by at least a factor of two, while 1,030
genes were repressed by a factor of two or more. Therefore, it was
found that a diauxic shift involves a staggering amount of gene
regulation. Of the 6,200 yeast genes, 1,740, or roughly 28%, appeared to be regulated as a result of a diauxic shift.
© 2010 The McGraw-Hill Companies, S.r.l. - Publishing Group Italia
Robert J. Brooker - Genetica
Because the gene sequence in each spot was known, the next
step was to relate the levels of gene expression based on the microarray data to specific genes. A common goal is to identify genes
whose pattern of expression seems to strongly correlate with each
other. This statistical technique is called a cluster analysis. The
graphs shown in the data of Figure EG21.1.1 illustrate how microarray data can be used to make this type of comparison. Though
the sequences of these genes were known from the yeast genomesequencing project, the functions of some of these genes were not
known. Each time point involved the measurement of the red:green
ratio at a particular spot. Figure EG21.1.1 shows the analysis of 28
genes (seven genes in each panel) over the course of 9 to 21 hours
following the addition of glucose. The diauxic shift occurred at approximately 15 hours.
Let’s examine the data shown in Figure EG21.1.1. In part (a),
the genes were induced at 15 to 17 hours of growth. Due to their
coordinate regulation, these genes may be controlled by the same
transcription factor(s) and may participate in a common metabolic
response to the induction of the TCA cycle. In fact, several of the
genes in part (a) have already been studied, and they are known to
play a role in the ability of the mitochondria to run the TCA cycle.
This makes sense because the diauxic shift occurred around 15
hours of growth, which correlates with the time when the genes
were induced. By comparison, the genes in part (b) were induced
later, after the TCA cycle had operated for a few hours and when
the carbon sources were becoming depleted. Therefore, it would
seem that these genes were induced as a response to the operation
of the TCA cycle or they were induced because the carbon
sources in the media were low. Why is this information useful?
Though further work needs to be done to elucidate the functions
of some of the genes shown in parts (a) and (b), the data suggest a
common regulation and metabolic function for particular groups
of genes. It would seem more likely that the transcription factor(s) that regulate the genes in part (a) would be different from
those that regulate the genes in part (b). Likewise, the proteins
encoded by these genes may work in different cellular pathways.
The genes shown in parts (c) and (d) illustrate a similar phenomenon, except that the switch to the TCA cycle and the operation of the TCA cycle represses those genes. The genes shown in
part (c) were active during glycolysis and then were repressed at
the time of the diauxic shift, while those shown in part (d) were
repressed after the TCA cycle had operated for a few hours. Most
of the genes shown in part (d) were already known to function as
ribosomal proteins. Therefore, as the carbon sources in the media
were depleted, these results suggest that one of the cellular responses is to diminish the synthesis of ribosomes, which, in turn,
would slow down the rate of protein synthesis. It would seem that
the yeast cells were trying to conserve energy at this late stage of
growth.
Overall, the data shown in the experiment of Figure EG21.1.1
illustrate how a microarray analysis can shed light on gene function at the genomic level. It provides great insight regarding gene
regulation and may help to identify groups of proteins (i.e., clusters) that share a common cellular function.
© 2010 The McGraw-Hill Companies, S.r.l. - Publishing Group Italia