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The budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe have been at the fore of genetic dissection. Indeed an acronym has been coined in their honour—TAPOYG, “the awesome power of yeast genetics.” One relatively straightforward but important screen was used to identify mutants that interfered with the cell-division cycle (CDC mutants), for which Leland Hartwell and Paul Nurse received Nobel prizes. Figure 16-9 depicts the kinds of mutations recovered in one screen, looking for mutations that block the mitotic cell cycle at specific points. 

Because many such mutations are expected to be lethal, the screen was for conditional heat-sensitive CDC mutations, which are wild type at low temperatures but mutant at high. They result from amino acid changes that lead to deleterious protein shape changes at high temperature. These mutants can be propagated at room temperature (the permissive temperature) and then shifted to high temperature (the restrictive temperature), at which they express the mutant phenotype. 

The set of mutants derived from this type of screen has enabled researchers to define many of the proteins that regulate the highly programmed progression through the cell cycle. Comparative genomics has shown that these same genes are at work in the cell cycle of humans and that many of these genes are defective in cancers.

Aspergillus is a filamentous fungus that like Neurospora has been an important genetic model organism. One interesting screen using Aspergillus was a visual screen for mutants with altered nuclear division. The screen revealed three main classes of mutants, nim (never in mitosis), bim (blocked in mitosis), and (nuclear distribution), as shown in Figure 16-10. As was also true for the yeast CDC mutants, these were heat-sensitive alleles that could be grown at the permissive temperature but shifted to restrictive temperature for the study of the phenotype. 

Subsequent studies showed that NimA is a kinase (phosphorylates other proteins), BimC is a kinesin (a motor; a protein that moves organelles on the cytoskeleton), and NudA is a dynein subunit (another motor). Thus these proteins are revealed as key players in the cell-division and growth process.

E. coli has several systems that make it ideal for genetic dissection. Foremost is the lacZ gene. The function of this gene (for the enzyme  galactosidase) can be conveniently assayed by adding a compound called X-gal to the medium. The LacZ protein converts X-gal to a blue colour (which happens to be the same dye that is used to stain blue jeans). 

The power of this system is that lacZ can be fused to genes of other proteins of interest; then its blue dye production acts as a reporter for that gene. Two types of fusions are possible, transcriptional fusions and translational fusions (Figure 16-11). Transcriptional fusions result in two separate proteins’ being made off one transcript. They are useful only for monitoring transcription levels because LacZ is made separately from the other protein. Translational fusions result in translation of a fused “hybrid” protein. They are useful for studies in which the hybrid protein (and hence the reporter LacZ) participate in the usual cellular transactions of the gene of interest. 

Translational fusions have been useful in the study of protein secretion out of an E. coli cell. Various secretory proteins such as membrane targeting proteins (signal sequences), membrane anchoring proteins, and secretory proteins have all been fused to LacZ. For example, when lacZ was transcriptionally fused to MalF, the gene for a membrane secretory protein, the accompanying GAL protein was thrust partway through the membrane and could not produce the blue colour, and so colonies were white. 

When this strain is mutagenized, mutants that affect any stage of the secretion process leave the GAL in the cytoplasm and a blue colony colour results. Hence, a screen for blue mutants revealed many mutants of interest. From such studies, the various players in the secretion process can be pieced together.

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