Figure 8–59 RNA interference provides a convenient method for conducting genome-wide genetic screens. In this experiment, each well in this 96-well plate is filled with E. coli that produce a different double-stranded RNA. Each interfering RNA matches the nucleotide sequence of a single C. elegans gene, thereby inactivating it. About 10 worms are added to each well, where they ingest the genetically modified bacteria. The plate is incubated for several days, which gives the RNAs time to inactivate their target genes—and the worms time to grow, mate, and produce offspring. The plate is then examined in a microscope, which can be controlled robotically, to screen for genes that affect the worms’ ability to survive, reproduce, develop, and behave. Shown here are normal worms alongside worms that show an impaired ability to reproduce due to inactivation of a particular “fertility” gene. (From B. Lehner et al., Nat. Genet. 38:896–903, 2006. With permission from Macmillan Publishers Ltd.) unknown reasons, RNAi does not efficiently inactivate all genes. Moreover, within whole organisms, certain tissues may be resistant to the action of RNAi (for example, neurons in nematodes). Another problem arises because many organisms contain large gene families, the members of which exhibit sequence similarity. RNAi therefore sometimes produces “off-target” effects, inactivating related genes in addition to the targeted gene. One strategy to avoid such problems is to use multiple small RNA molecules matched to different regions of the same gene. Ultimately, the results of any RNAi experiment must be viewed as a strong clue to, but not necessarily a proof of, normal gene function.
In the preceding section, we discussed how genetic approaches can be used to assess a gene’s function in cultured cells or, even better, in the intact organism. Although this information is crucial to understanding gene function, it does not generally reveal the molecular mechanisms through which the gene product works in the cell. For example, genetics on its own rarely tells us all the places in the organism where the gene is expressed, or how its expression is controlled. It does not necessarily reveal whether the gene acts in the nucleus, the cytosol, on the cell surface, or in one of the numerous other compartments of the cell. And it does not reveal how a gene product might change its location or its expression pattern when the external environment of the cell changes. Key insights into gene function can be obtained by simply observing when and where a gene is expressed. A variety of approaches, most involving some form of genetic engineering, can easily provide this critical information.
As discussed in detail in Chapter 7, cis-regulatory DNA sequences, located upstream or downstream of the coding region, control gene transcription. These regulatory sequences, which determine precisely when and where the gene is expressed, can be easily studied by placing a reporter gene under their control and introducing these recombinant DNA molecules into cells (Figure 8–60). In this way, the normal expression pattern of a gene can be determined, as well as 123 start site for RNA pattern of normal gene X
DNA sequences that determine the expression of gene X cells 123 pattern of reporter gene Y expression
CONCLUSIONS —cis-regulatory sequence 3 normally turns on gene X in cell B —cis-regulatory sequence 2 normally turns on gene X in cells D, E, and F —cis-regulatory sequence 1 normally turns off gene X in cell D
Figure 8–60 Using a reporter protein to determine the pattern of a gene’s expression. (A) In this example, the coding sequence for protein X is replaced by the coding sequence for reporter protein
Y. The expression patterns for X and Y are the same. (B) Various fragments of DNA containing candidate cis-regulatory sequences are added in combinations to produce test DNA molecules encoding reporter gene Y. These recombinant DNA molecules are then tested for expression after introducing them into a variety of different types of mammalian cells. The results are summarized in (C).