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▲ FIGURE 15-2 Evolutionary conservation of core processes in human, fruit fly (Drosophila), and roundworm (C. elegans) genomes. On the basis of fairly stringent criteria for protein similarity, humans, flies, and worms have in common about 1500 genes distributed among the functional classes shown in this pie chart. About 28 percent of this common set of genes encode proteins that function in signaling or gene control. The molecular functions of about one-third of the genes and proteins common to these species are not yet known. [Adapted from J. C. Venter et al., 2001, Science 291:1304.]

In Situ Hybridization Can Detect Transcription Changes in Intact Tissues and Permeabilized Embryos

A common effect of external signals is to alter the pattern of gene expression by a cell. Signal-induced changes in the expression of particular genes is usually monitored by measuring the corresponding mRNAs or proteins in the presence and absence of a signal. The total cellular mRNA can be extracted, separated by gel electrophoresis, and subjected to Northern blotting, which detects individual mRNAs by hybridization to labeled complementary DNA probes (see Figure 9-26). Likewise, cellular proteins can be extracted, separated elec-trophoretically, and subjected to Western blotting, a procedure in which individual proteins separated on the blot are detected with specific antibodies (see Figure 3-35). These blotting methods are generally not sensitive enough to determine changes within a single cell. The polymerase chain reaction (PCR), however, can amplify a specific mRNA from a single cell so that it is detectable (see Figure 9-24).

Both Northern blotting and PCR amplification require extracting the mRNA from a cell or mixture of cells, which means that the cells are removed from their normal location within an organism or tissue. As a result, the location of a cell and its relation to its neighbors is lost. To retain such positional information, a whole or sectioned tissue or even

▲ EXPERIMENTAL FIGURE 15-3 In situ hybridization can detect activity of specific genes in whole and sectioned embryos. The specimen is permeabilized by treatment with detergent and a protease to expose the mRNA to the probe. A DNA or RNA probe, specific for the mRNA of interest, is made with nucleotide analogs containing chemical groups that can be recognized by antibodies. After the permeabilized specimen has been incubated with the probe under conditions that promote hybridization, the excess probe is removed with a series of washes. The specimen is then incubated in a solution containing an antibody that binds to the probe. This antibody is covalently joined to a reporter enzyme (e.g., horseradish peroxidase or alkaline phosphatase) that produces a colored reaction product. After excess antibody has been removed, substrate for the a whole permeabilized embryo may be subjected to in situ hybridization to detect the mRNA encoded by a particular gene (Figure 15-3). This technique allows gene transcription to be monitored in both time and space. Immunohis-tochemistry, the related technique of staining tissue with fluorescence-labeled antibodies against a particular protein, provides similar information for proteins, an important advantage for obtaining ideas about protein function from its subcellular location (see Figures 5-33 and 5-45).

DNA Microarray Analysis Can Assess Expression of Multiple Genes Simultaneously

A major limitation of in situ hybridization and blotting techniques is that the mRNA or protein product of only a few genes can be examined at a time. Thus monitoring the activity of many genes by these methods requires multiple assays. In contrast, researchers can monitor the expression of thousands of genes at one time with DNA microarrays (see Figure 9-35). In this technique, cDNAs labeled with a fluorescent dye are made from the total mRNA extracted from the cells under study. The labeled cDNAs are then hybridized to a microscope slide dotted with spots of DNA. Each DNA spot contains a unique sequence from a particular gene, and tens of thousands of genes can be represented on a standard slide. The fluorescence of spots that reporter enzyme is added. A colored precipitate forms where the probe has hybridized to the mRNA being detected. (a) A whole mouse embryo at about 10 days of development probed for Sonic hedgehog mRNA. The stain marks the notochord (red arrow), a rod of mesoderm running along the future spinal cord. (b) A section of a mouse embryo similar to that in part (a). The dorsal/ventral axis of the neural tube (NT) can be seen, with the Sonic hedgehog-expressing notochord (red arrow) below it and the endoderm (blue arrow) still farther ventral. (c) A whole Drosophila embryo probed for an mRNA produced during trachea development. The repeating pattern of body segments is visible. Anterior (head) is up; ventral is to the left. [Courtesy of L. Milenkovic and M. P Scott.]

▲ EXPERIMENTAL FIGURE 15-3 In situ hybridization can detect activity of specific genes in whole and sectioned embryos. The specimen is permeabilized by treatment with detergent and a protease to expose the mRNA to the probe. A DNA or RNA probe, specific for the mRNA of interest, is made with nucleotide analogs containing chemical groups that can be recognized by antibodies. After the permeabilized specimen has been incubated with the probe under conditions that promote hybridization, the excess probe is removed with a series of washes. The specimen is then incubated in a solution containing an antibody that binds to the probe. This antibody is covalently joined to a reporter enzyme (e.g., horseradish peroxidase or alkaline phosphatase) that produces a colored reaction product. After excess antibody has been removed, substrate for the reporter enzyme is added. A colored precipitate forms where the probe has hybridized to the mRNA being detected. (a) A whole mouse embryo at about 10 days of development probed for Sonic hedgehog mRNA. The stain marks the notochord (red arrow), a rod of mesoderm running along the future spinal cord. (b) A section of a mouse embryo similar to that in part (a). The dorsal/ventral axis of the neural tube (NT) can be seen, with the Sonic hedgehog-expressing notochord (red arrow) below it and the endoderm (blue arrow) still farther ventral. (c) A whole Drosophila embryo probed for an mRNA produced during trachea development. The repeating pattern of body segments is visible. Anterior (head) is up; ventral is to the left. [Courtesy of L. Milenkovic and M. P Scott.]

hybridize to a cDNA species is measured with an instrument that scans the slide. Fluorescing spots thus represent active genes, which have been transcribed into their mRNAs (see Figure 1-23).

Microarray experiments are commonly used to compare the mRNAs produced by two different populations of cells: for example, two distinct cell types, the same cell type before and after some treatment, or mutant and normal cells. An example of a microarray-based discovery comes from the results of studies of cultured fibroblast cells, which have long been known to initiate cell division when serum containing growth factors is added to the medium. Microarray analysis of gene expression at different times after treatment of fibroblasts with serum showed that transcription of about 500 of the 8613 genes examined changed substantially over time (see Figure 9-36). Transcriptional changes were detected within 15 minutes, with genes encoding proteins that control progression through the cell cycle becoming active first. Later, genes encoding proteins with roles in wound healing, such as clotting factors and attractants for immune-system cells, became active. The production of these proteins suggests that proliferating fibroblasts are stimulated by serum to participate in wound healing, something that had not been known. In retrospect, it makes sense, because the time during which fibroblasts are exposed to serum in an intact organism is when there is a wound. The results show the usefulness of microarrays in revealing unexpected responses by cells.

The developmental time course of gene transcription has been assessed with DNA microarrays for the nematode C. elegans and the fly Drosophila. In recent experiments, microarrays representing about 94 percent of the C. elegans genes were used to monitor transcription at different stages of development and in both sexes. The results showed that expression of about 58 percent of the monitored genes changes more than twofold during development, and another 12 percent are transcribed in sex-specific patterns. Findings from a similar study assessing about one-third of all Drosophila genes showed that transcription of more than 90 percent of them changes by twofold or more during development and that most genes are used repeatedly during development (Figure 15-5). These results clearly show that development is marked by extensive changes in transcription, with few genes exhibiting a monotonous pattern of unchanging transcription.

HIn the future, microarray analysis will be a powerful diagnostic tool in medicine. For instance, particular sets of mRNAs have been found to distinguish tumors with a poor prognosis from those with a good prognosis (Chapter 23). Previously indistinguishable disease variations are now detectable. Analysis of tumor biopsies for these distinguishing mRNAs will help physicians to select the most appropriate treatment. As more patterns of gene expression characteristic of various diseased tissues are recognized, the diagnostic use of DNA microarrays will be extended to other conditions. I

Protein Microarrays Are Promising Tools for Monitoring Cell Responses That Include Changes in Protein-Binding Patterns

A cell's response to signals can include not only changes in gene expression, but also alterations in the modifications of proteins and the associations between proteins. As discussed in other chapters, the activities of many proteins depend on their association with other proteins or with small intracellular signaling molecules (e.g., cAMP or phosphoinositides). Two common examples are the activation of adenylyl cyclase by interaction with Gsa • GTP (see Figure 13-11) and the activation of protein kinase A by binding of cAMP (see Figure 3-27). The activity of some transcriptional regulators (e.g., CREB) also depend on their associating with another protein (see Figure 13-32). The results of systematic studies are beginning to reveal protein-protein associations that are critical for cell functioning and how these associations change in response to signals. For example, scientists have produced large quantities of 5800 yeast proteins (=80 percent of the total proteins) by cloning them in high-level expression vectors in yeast and purifying the individual proteins. In a technique analogous to DNA microarrays, small samples of the purified yeast proteins can be spotted on microscope slides to produce a protein microarray, also called a pro-teome chip.

To test the efficacy of assaying protein-protein associations on such arrays, researchers exposed the yeast protein microarray to biotin-labeled calmodulin, a calcium-binding protein. After excess calmodulin was removed from the microarray, binding of calmodulin to proteins in the array was detected with a fluorescent reagent specific for biotin (Figure 15-4). This experiment succeeded in detecting six proteins already known to bind calmodulin. Six other known calmodulin-binding proteins were not detected, two because they were not included in the array and four that may have been underproduced. In principle, others could be missed because proteins associate only as part of a complex of more than two proteins or because the protein fastened to the chip is in the wrong conformation for binding. Despite these possible problems, 33 other calmodulin-binding yeast proteins not previously recognized also were detected. The gene sequences corresponding to the 39 calmodulin-binding proteins detected indicate that 14 of these proteins have a common motif that may form the binding surface. The results of such experiments show that protein arrays will be a useful, if not completely comprehensive and accurate, tool for monitoring associations of proteins as indicators of cell responses.

Systematic Gene Inactivation by RNA Interference

Changes in transcription at various developmental stages provide one criterion for identifying genes that play a critical role in cell regulation and differentiation. A more important

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