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9.11: Cell-specific gene expression

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    4893
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    Figure 9.11.2 Even-skipped gene courtesy of Peter A. Lawrence and Blackwell Scientific Publications

    An example: Make Drosophila transgenic for recombinant DNA containing:

    • the Z gene for beta-galactosidase
    • even-skipped (eve), (another homeobox gene).

    Any cell with the transcription factors for turning on the even-skipped promoter will begin to make beta-galactosidase. Given the proper substrate, the enzyme produces a colored product.

    The above photomicrograph shows 7 bands of this colored product identifying the cells that were expressing the even-skipped gene. This event was "reported" by the lacZ gene. The 7 dark stripes reveal regions that alternate with the 7 bands formed by the cells expressing fushi-tarazu.

    Green fluorescent protein (GFP)

    In nature, green fluorescent protein (GFP) is produced by, Aequorea victoria, the Pacific Northwest jellyfish. The protein has become of great interest to cell and molecular biologists because it can reveal gene expression in living cells.

    This is done by fusing the gene for GFP to the gene whose expression you are interested in. When that gene is turned on in a cell, not only is its protein synthesized, but GFP is synthesized as well. Illuminating the cells with near-ultraviolet light causes them to fluoresce a bright green. In this way, the experimenter can see when and where the gene is expressed in the living organism.

    DNA Chips

    All the methods described so far are limited to monitoring the expression of one or, at most, a few genes. But as conditions change in a cell, the transcription and translation of literally hundreds of genes may be altered.

    Thanks to the marriage of

    • semiconductor chip technology
    • automated synthesis of oligodeoxynucleotides
    • automated fluorescence scanners
    • computer software,

    it is now possible to monitor the activity of literally thousands of genes in one kind of cell. For examples:

    • mammalian cells when they are transferred from a "minimal" culture medium to one enriched in growth factors;
    • the skeletal muscles of mice as they age.

    The Chip

    alt
    Figure 9.11.3 DNA chip
    • Examine published gene sequences.
    • For each gene, pick out ~20 different stretches of ~25 nucleotides that seem characteristic of that gene.
    • Synthesize oligodeoxynucleotides corresponding to these.
    • Also synthesize oligodeoxynucleotides for each of the above that have one nucleotide altered (usually near the middle). These will provide a control.
    • Using robotic chip-making machines, spot these oligonucleotides individually in arrays, each spot receiving millions of copies that are fixed to the chip surface.

    With the partial completion of the human genome project, three companies are now selling DNA chips containing from 36,000 to 50,000 pieces of DNA thought to represent different human genes.

    The Assay

    • Harvest your cells. Presumably they are expressing a characteristic subset of their genes; that is, transcribing them into messenger RNA (mRNA) molecules.
    • Extract the RNA.
    • Make complementary DNA (cDNA) by treating the RNA mixture with reverse transcriptase.
    • Transcribe the cDNA back into now much-amplified RNA.
    • Attach fluorescent tags to the RNA.
    • Flood the chip with this mixture.
    • RNAs finding their complementary sequences on the chip will bind to them. (They will bind less strongly to adjacent spots with the single-nucleotide change if the binding is truly specific.
    • Illuminate the chip and automatically record the intensities of the color at each spot.
    • Use a computer to analyze the pattern.

    Results of monitoring genome-wide expression

    • This work was reported by V. R. Iyer, et al in the 1 January 1999 issue of Science. It involved the monitoring the expression of 8613 different genes.
    • Mice raised on a restricted diet did not show such dramatic shifts in gene expression as they aged. This fits well with data that mice on restricted diets age more slowly than those on rich diets.

    This page titled 9.11: Cell-specific gene expression is shared under a CC BY 3.0 license and was authored, remixed, and/or curated by John W. Kimball via source content that was edited to the style and standards of the LibreTexts platform; a detailed edit history is available upon request.

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