ESSENTIAL

CELL BIOLOGY
ESSENTIAL
third edition

CELL BIOLOGY

third edition
Alberts Bray Hopkin Johnson Lewis Raff Roberts Walter

ISBN 978-0-8153-4130-7 Alberts Bray Hopkin Johnson Lewis Raff Roberts Walter

9 780815 341307 ECB3 interactive DVD-ROM inside

352 Chapter 10 analyzing Genes and Genomes

protein strategy has become a standard way to determine the distribution

and to track the movement of any protein of interest in a living organism.
From this information, many insights about a protein’s function in the
organism can be obtained.
It is also possible to directly observe the time and place that the mRNA
product of a gene is expressed. In most cases, this strategy provides the
same overall information as the reporter gene approach. In some situa-
tions, however, monitoring RNA is the method of choice: for example, if
the gene’s final product is RNA rather than protein. The technique, which
relies on the principles of nucleic acid hybridization described earlier, is
called in situ hybridization (from the Latin in situ, “in place”), because it
allows specific nucleic acid sequences to be located while they are still in
place within cells or within chromosomes.
In situ hybridization uses nucleic acid probes, labeled with either fluores-
cent dyes or radioactive isotopes, to detect the presence of RNA or DNA
of a particular sequence within a cell or tissue (Figure 10–31). This tech-
nique can reveal gene expression patterns, and has led to great advances
in our understanding of embryonic development, by making easily visible
the many changes in gene expression that occur in different cells of the
developing embryo. In situ hybridization also allows the visualization of
specific DNA sequences within chromosomes. In this case the probes are
hybridized to whole chromosomes that have been exposed briefly to a
very high pH to separate the two DNA strands. The chromosomal regions
that bind the labeled probe can then be seen (Figure 10–32). This tech-
nique can be used medically to identify, early in a pregnancy, fetuses that
carry abnormal chromosomes.
Figure 10–30 Green fluorescent protein
(GFP) can be used to identify specific
cells in a living animal. For this experiment, Hybridization on DNA Microarrays Monitors the
carried out in the fruit fly, the GFp gene Expression of Thousands of Genes at Once
was joined (using recombinant DNa
techniques) to a fly promoter that is active We saw in Chapter 8 that a cell expresses only a subset of the genes
only in a specialized set of neurons. this available in its genome. One of the most important uses of nucleic acid
image of a live fly embryo was captured
hybridization is to determine, for a population of cells, exactly which genes
by a fluorescence microscope and shows
approximately 20 neurons, each with long are actively being transcribed into mRNA and which are transcription-
ECB3(axons
projections e10.35/10.30
and dendrites) that ally silent. In situ hybridization methods allow scientists to monitor the
communicate with other (nonfluorescent) expression of one gene—or relatively few genes—at a time. In the 1990s,
cells. these neurons, located just under the however, investigators developed a new tool, called a DNA microarray,
embryo’s surface, allow the organism to
that allows the RNA products of tens of thousands of genes to be moni-
sense its immediate environment.
(From W.B. Grueber et al., Curr. Biol. tored at the same time. By examining the expression of so many genes
13:618–626, 2003. With permisison from simultaneously, we can begin to identify and study the complex gene
elsevier.) expression patterns that underlie cellular physiology, visualizing which
genes are switched on (or off) as cells grow, divide, or respond to hor-
mones, toxins, or infection.
DNA microarrays are little more than glass microscope slides studded
with a large number of DNA fragments, each containing a nucleotide
sequence that serves as a probe for a specific gene. The most dense arrays
contain hundreds of thousands of these fragments in an area smaller than
a postage stamp, allowing the expression patterns of entire genomes to

Figure 10–31 In situ hybridization can be used to detect the

presence of a virus in cells. In this micrograph, the nuclei of epithelial
cells infected with the human papillomavirus (hpV) are stained pink
by a probe that recognizes a viral DNa sequence. the nuclei of all
cells are stained blue, although this is masked by the pink stain in the
infected cells. the cytoplasm of all cells is stained green. (Courtesy of
50 mm hogne røed Nilsen.)
 Deciphering and exploiting Genetic Information 353

Figure 10–32 In situ hybridization is used to locate genes on

chromosomes. here, six different DNa probes have been used
to mark the locations of their respective nucleotide sequences on
human Chromosome 5 isolated in the metaphase stage of mitosis
(see Figure 5–16 and panel 18–1, pp. 626–627). the DNa probes
have been chemically labeled and are detected using fluorescent
antibodies specific for the chemical label. Both the maternal and
paternal copies of Chromosome 5 are shown, aligned side by side.
each probe produces two dots on each chromosome because
chromosomes undergoing mitosis have already replicated their DNa
and therefore each chromosome contains two identical DNa helices.
the technique employed here is nicknamed FISh, for fluorescence in
situ hybridization. (Courtesy of David C. Ward.)

2 mm

be monitored in a single experiment. Some types of microarrays carry

DNA fragments corresponding to entire genes that are spotted onto the
slides by a robot. Other types contain short, single-stranded DNA mol-
ecules that are synthesized on the surface of the wafer with techniques
similar to those that are used to etch circuits onto computer chips. In
either case, the exact sequence—and position—of every DNA probe on
the chip is known.
To use a DNA microarray to monitor the expression of every gene in a
cell simultaneously, mRNA from the cells being studied is extracted and
converted to cDNA (see Figure 10–13). The cDNA is then labeled with a
fluorescent probe. The microarray is incubated with the labeled cDNA
mRNA from mRNA from
sample, and hybridization is allowed to occur (Figure 10–33). The array is sample 1 sample 2
then washed to remove unbound molecules, and the positions to which
fluorescently labeled DNA fragments have hybridized, through comple- ECB3 E10.16/10.32
mentary base-pairing, are identified by an automated microscope. The convert to cDNA, convert to cDNA,
array positions are then matched to the particular genes whose DNA was label with red label with green
fluorochrome fluorochrome
originally spotted at each location.
DNA microarrays have been used to examine everything from the
changes in gene expression that make strawberries ripen to the genetic
‘signatures’ of different types of human cancer cells. Comparisons of the
gene expression profiles of human cancers, for example, can be used to
readily distinguish one type of cancer cell from another. By relating these
HYBRIDIZE TO MICROARRAY
expression patterns to clinical data gathered for each cancer—including
how rapidly it progresses and whether it responds to treatment—it may
be possible to predict whether a particular patient will respond to a spe-
cific therapy. Thus microarray-based ‘profiles’ of cancer cells are likely to
lead to much more precise and effective treatments for this often fatal
disease. WASH; SCAN RED AND GREEN SIGNALS
AND COMBINE IMAGES

Figure 10–33 DNA microarrays are used to monitor the expression

of many thousands of genes simultaneously. In this example, mrNa
is collected from two different cell samples for direct comparison of
their relative levels of gene expression—for example, cells treated
with a hormone and untreated cells of the same type. the samples are
converted to cDNa and labeled, one with a red fluorescent dye, the
other with a green fluorescent dye. the labeled samples are mixed
and then allowed to hybridize to the microarray. after incubation, the
array is washed and the fluorescence scanned. Only a small proportion
of the microarray, representing 110 genes, is shown. Red spots
indicate that the gene in sample 1 is expressed at a higher level than
the corresponding gene in sample 2, and green spots indicate that
expression of the gene is more vigorous in sample 2 than in sample 1.
Yellow spots reveal genes that are expressed at equal levels in both
cell samples. Dark spots indicate little or no expression of the gene small region of microarray
whose fragment is located at that position in the array. representing 110 genes