From Genomes to Proteomes:- Cellular Expression Patterns Can Reveal the Cellular Function of a Gene

In every newly sequenced genome, researchers find genes that encode proteins with no evident structural relationships to known genes or proteins. In these cases, other approaches must be used to generate information about gene function. Determining which tissues a gene is expressed in, or what circumstances trigger the appearance of the gene product, can provide valuable clues. Many different approaches have been developed to study these patterns.

Two-Dimensional Gel Electrophoresis As shown in Figure 3–22, two-dimensional gel electrophoresis allows the separation and display of up to 1,000 different proteins on a single gel. Mass spectrometry can then be used to partially sequence individual protein spots and assign each to a gene. The appearance and nonappearance (or disappearance) of particular protein spots in samples from different tissues, from similar tis sues at different stages of development, or from tissues treated in ways that simulate a variety of biological conditions can help define cellular function.

DNA Microarrays Major refinements of the technology underlying DNA libraries, PCR, and hybridization have come together in the development of DNA microar rays (sometimes called DNA chips), which allow the rapid and simultaneous screening of many thousands of genes. DNA segments from known genes, a few dozen to hundreds of nucleotides long, are amplified by PCR and placed on a solid surface, using robotic devices that accurately deposit nanoliter quantities of DNA solution. Many thousands of such spots are deposited in a predesigned array on a surface area of just a few square centimeters. An alternative strategy is to synthesize DNA directly on the solid surface, using photolithography (Fig. 9–21). Once the chip is constructed, it can be probed with mRNAs or cDNAs from a particular cell type or cell culture to identify the genes being expressed in those cells.

FIGURE 9–21 Photolithography. This technique for preparing a DNA microarray makes use of nucleotide precursors that are activated by light, joining one nucleotide to the next in a photoreaction (as op posed to the chemical process illustrated in Fig. 8–38). A computer is programmed with the oligonucleotide sequences to be synthesized at each point on a solid surface. The surface is washed successively with solutions containing one type of activated nucleotide (A*, G*, etc.). As in the chemical synthesis of DNA, the activated nucleotides are blocked so that only one can be added to a chain in each cycle. A screen covering the surface is opened over the areas programmed to receive a particular nucleotide, and a flash of light joins the nucleotide to the polymers in the uncovered areas. This continues until the re quired sequences are built up on each spot on the surface. Many polymers with the same sequence are generated on each spot, not just the single polymer shown. Also, the surfaces have thousands of spots with different sequences (see Fig. 9–22); this array shows just four spots, to illustrate the strategy.

A microarray can answer such questions as which genes are expressed at a given stage in the development of an organism. The total complement of mRNA is isolated from cells at two different stages of development and converted to cDNA, using reverse transcriptase and fluorescently labeled deoxynucleotides. The fluorescent cDNAs are then mixed and used as probes, each hybridizing to complementary sequences on the microarray. In Figure 9–22, for example, the labeled nucleotides used to make the cDNA for each sample fluoresce in two different colors. The cDNA from the two samples is mixed and used to probe the microarray. Spots that fluoresce green represent mRNAs more abundant at the single-cell stage; those that fluoresce red represent sequences more abundant later in development. The mRNAs that are equally abundant at both stages of development fluoresce yellow. By using a mixture of two samples to measure relative rather than absolute abundance of sequences, the method corrects for variations in the amounts of DNA originally deposited in each spot on the grid and other possible inconsistencies among spots in the microarray. The spots that fluoresce provide a snapshot of all the genes being expressed in the cells at the moment they were harvested—gene ex pression examined on a genome-wide scale. For a gene of unknown function, the time and circumstances of its expression can provide important clues about its role in the cell.

An example of this technique is illustrated in Fig ure 9–23, showing the dramatic results this technique can produce. Segments from each of the more than 6,000 genes in the completely sequenced yeast genome were separately amplified by PCR, and each segment was deposited in a defined pattern to create the illustrated microarray. In a sense, this array provides a snap shot of the entire yeast genome.

Protein Chips Proteins, too, can be immobilized on a solid surface and used to help define the presence or absence of other proteins in a sample. For example, re searchers prepare an array of antibodies to particular proteins by immobilizing them as individual spots on a solid surface. A sample of proteins is added, and if the protein that binds any of the antibodies is present in the sample, it can be detected by a solid-state form of the ELISA assay. Many other types and ap plications of protein chips are being developed.

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مواضيع ذات صلة

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