Figure 8–57 Genome-wide screens for fitness using a large pool of barcoded yeast deletion mutants. A large pool of yeast mutants, each with a different gene deleted and present in equal amounts, is grown under conditions selected by the experimenter. Some mutants (blue) grow normally, but others show reduced growth (orange and green) or no growth at all (red). The fitness of each mutant is experimentally determined in the following way. After the growth phase is completed, genomic DNA (isolated from the mixture of strains) is purified and the relative abundance of each mutant is determined by quantifying the level of the DNA barcode matched to each deletion. This can be done by sequencing the pooled genomic DNA or hybridizing it to microarrays (see Figure 8–64) that contain DNA oligonucleotides complementary to each barcode. In this way, the contribution of every gene to growth under the specified condition can be rapidly ascertained. This type of study has revealed that of the approximately 6000 coding genes in yeast, only about 1000 are essential under standard growth conditions.
In S. cerevisiae, the task of generating a complete set of 6000 mutants, each missing only one gene, was accomplished several years ago. Because each mutant strain has an individual barcode sequence embedded in its genome, a large mixture of engineered strains can be grown under various selective test conditions— such as nutritional deprivation, a temperature shift, or the presence of various drugs—and the cells that survive can be rapidly identified by the unique sequence tags present in their genomes. By assessing how well each mutant in the mixture fares, one can begin to discern which genes are essential, useful, or irrelevant for growth under the various conditions (Figure 8–57).
The insights generated by examining mutant libraries can be considerable. For example, studies of an extensive collection of mutants in Mycoplasma genitalium—the organism with the smallest known genome—have identified the minimum complement of genes essential for cellular life. Growth under laboratory conditions requires about three-quarters of the 480 protein-coding genes in
M. genitalium. Approximately 100 of these essential genes are of unknown function, which suggests that a surprising number of the basic molecular mechanisms that underlie life have yet to be discovered.
Collections of mutant organisms are also available for many animal and plant species. For example, it is possible to “order,” by phone or email from a consortium of investigators, a deletion or insertion mutant for almost all coding genes in Drosophila. Likewise, a nearly complete set of mutants exists for the “model” plant Arabidopsis. And the adaptation of the CRISPR system for use in mice means that, in the near future, we can expect to be able to turn on or off—at will—each gene in the mouse genome. Although we are still ignorant about the function of most genes in most organisms, these technologies allow an exploration of gene function on a scale that was unimaginable a decade ago.
RNA Interference Is a Simple and Rapid Way to Test Gene Function
Although knocking out (or conditionally expressing) a gene in an organism and studying the consequences is the most powerful approach for understanding the functions of the gene, RNA interference (RNAi, for short), is an alternative, particularly convenient approach. As discussed in Chapter 7, this method exploits a natural mechanism used in many plants, animals, and fungi to protect themselves against viruses and transposable elements. The technique introduces into a cell or organism a double-stranded RNA molecule whose nucleotide sequence matches that of part of the gene to be inactivated. After the RNA is processed, it hybridizes with the target-gene RNA (either mRNA or noncoding RNA) and reduces its expression by the mechanisms shown in Figure 7–75.