Bypass and interaction suppressors; pathway analysisThe isolation of extragenic suppressors is a powerful tool for identifyinggenes that encode proteins that function in the same process as a gene ofinterest.Bypass suppressors suppress all mutant alleles of a gene, including nullalleles, and hence bypass the requirement for a gene. These suppressorsare gene specific, allele nonspecific. There are three types of bypasssuppressors that we will consider here.Often bypass suppressors can be generated by up-regulation of agene in a parallel pathway. One good example of bypass suppression byup-regulation of a parallel pathway is suppression of yeast cyc1 loss-of-function mutants by mutations that increase the expression of the CYC7gene. CYC1 encodes the major form of cytochrome c, and CYC7 encodes aminor form. The mutant phenotype of CYC1 can be suppressed by amutation in CYC7 that increases its expression. This mutation is causedby the transposable element Ty1 inserting into the promoter of the CYC7gene.Bypass suppressors can also result by altering the function of a gene thatacts in a distinct process. Bypass suppressors of mutations in the E. colimaltose permease gene, which encodes a protein that transports maltoseinto the cell, alter the specificity of the lactose permease gene. Lactosepermease normally transports lactose into the cell, but the bypasssuppressors now cause it to transport maltose.Epistatic interactionsBypass suppressors can also lie in genes that act downstream in aregulatory pathway, and in this case can referred to as epistaticsuppressors. (Some do not consider epistatic interactions to besuppression. This is a semantic issue that we won’t worry about, and wewill consider an epistatic suppressor to be a type of bypass suppressor.)Positive regulationLet's consider an example of epistatic interactions between genes that areinvolved in determining sex in the nematode C. elegans. As in Drosophila,the X chromosome to autosome ratio determines sex in C. elegans. Theratio is 1.0 in XX animals, causing them to develop as self-fertilizinghermaphrodites (hermaphrodites are basically females that producesperm for a short time). The ratio is 0.5 in X0 animals (there is no Ychromosome in C. elegans), causing them to develop as males. The tra-1and tra-2 genes are part of a pathway that determines sex in C. elegans. Inthis pathway, tra-2 encodes a cell surface receptor that is upstream of tra-1,which encodes a transcription factor that regulates genes involved insexual differentiation. In XX animals, the X to autosome signal activatesthe pathway that contains the tra-2 and tra-1 genes to produce thehermaphrodite fate. Loss-of-function mutations in tra-2 or tra-1 transformXX animals into males. The transformation by tra-2 mutations can besuppressed by certain mutations in the tra-1 gene: XX animals that aredoubly mutant for a loss-of-function tra-2 mutation and a gain-of-function tra-1 mutation develop as hermaphrodites.As with the CYC7 suppressors described above, the tra-1suppressors bypass the requirement for another gene. The difference isthat tra-2 and tra-1 act in the same pathway, whereas CYC1 and CYC7 actin parallel pathways. How can you distinguish between bypasssuppressors in the same or in parallel pathways. Analysis of double loss-of-function mutants can sometimes be used to distinguish between thetwo. In the case of parallel pathways, the double mutants will result in amore severe phenotype. In the cyc1; cyc2 double mutant, for example, nocytochrome c is produced, whereas some cytochrome c is produced ineither single loss-of-function mutant. The tra-2; tra-1 double mutant, bycontrast, has the identical phenotype to either single mutant.Negative regulationA third example of bypass suppression is in a pathway containing anegative regulatory interaction. In this case a mutation in a negativeregulatory gene produces a phenotype because the gene downstream isactivated inappropriately. But a loss-of-function mutation in thedownstream gene suppresses this phenotype. It is worth noting thatinactivating the downstream gene usually leads not only to suppression,but to an additional and often opposite phenotype. One good example ofthis type of suppression is in C. elegans sexual development. One of thegenes that the transcription factor TRA-1 negatively regulates in XXanimals is the cell death gene egl-1, which is required for programmed celldeath in C. elegans. A sexually dimorphic cell in C. elegans is the HSNmotor neuron, which innervates vulval muscles and stimulates egg layingby hermaphrodites. Males don't lay eggs and hence don't need HSNs; theHSNs undergo programmed cell death in males. In XX animals lackingtra-1 function, the HSNs adopt the male fate and die because the cell deathgene egl-1 is expressed and activates the cell death pathway. Buteliminating egl-1 function in a tra-1 XX animal suppresses the cell deathphenotype caused by the tra-1 mutation. This suppression occurs only forthe HSNs. Cells other than the HSNs still adopt male fates in XX animalscontaining mutations in tra-1 and egl-1 because TRA-1 regulates othergenes in these cells.Interaction suppressors are compensating mutations in physicallyinteracting components. A mutation in one protein disrupts theinteraction, but a mutation in the second protein restores the interaction.These mutations are gene specific and allele specific.The actin and fimbrin proteins inteact in budding yeast, and thisinteraction is essential for viability. Many proteins bind to actin to regulateactin assembly in cells. In order to find proteins that regulate actinassembly in vivo, suppressors of a temperature-sensitive allele of the yeastactin gene, ACT1, were isolated. A diploid homozygous act1/act1 mutantwas mutagenized, and hence only dominant suppressors were isolated.The investigators assumed that the original actin mutation impaired theinteraction of an actin-binding protein with actin. They reasoned thatisolation of dominant suppressors was more likely to yield mutations ingenes that encode interacting proteins because such mutants would bemutations that restored interactions that were disrupted by the actinmutation. Some suppressors were in the gene SAC6, which was latershown to encode the actin-binding protein fimbrin. When the sac6mutations were separated from the act1 mutation, it was found that thesac6 mutants were also inviable at higher temperatures. Thus, the twomutants, act1 and sac6, were on their
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