Bypass and interaction suppressors pathway analysis The isolation of extragenic suppressors is a powerful tool for identifying genes that encode proteins that function in the same process as a gene of interest Bypass suppressors suppress all mutant alleles of a gene including null alleles and hence bypass the requirement for a gene These suppressors are gene specific allele nonspecific There are three types of bypass suppressors that we will consider here Often bypass suppressors can be generated by up regulation of a gene in a parallel pathway One good example of bypass suppression by up regulation of a parallel pathway is suppression of yeast cyc1 loss offunction mutants by mutations that increase the expression of the CYC7 gene CYC1 encodes the major form of cytochrome c and CYC7 encodes a minor form The mutant phenotype of CYC1 can be suppressed by a mutation in CYC7 that increases its expression This mutation is caused by the transposable element Ty1 inserting into the promoter of the CYC7 gene Bypass suppressors can also result by altering the function of a gene that acts in a distinct process Bypass suppressors of mutations in the E coli maltose permease gene which encodes a protein that transports maltose into the cell alter the specificity of the lactose permease gene Lactose permease normally transports lactose into the cell but the bypass suppressors now cause it to transport maltose Epistatic interactions Bypass suppressors can also lie in genes that act downstream in a regulatory pathway and in this case can referred to as epistatic suppressors Some do not consider epistatic interactions to be suppression This is a semantic issue that we won t worry about and we will consider an epistatic suppressor to be a type of bypass suppressor Positive regulation Let s consider an example of epistatic interactions between genes that are involved in determining sex in the nematode C elegans As in Drosophila the X chromosome to autosome ratio determines sex in C elegans The ratio is 1 0 in XX animals causing them to develop as self fertilizing hermaphrodites hermaphrodites are basically females that produce sperm for a short time The ratio is 0 5 in X0 animals there is no Y chromosome in C elegans causing them to develop as males The tra 1 and tra 2 genes are part of a pathway that determines sex in C elegans In this pathway tra 2 encodes a cell surface receptor that is upstream of tra 1 which encodes a transcription factor that regulates genes involved in sexual differentiation In XX animals the X to autosome signal activates the pathway that contains the tra 2 and tra 1 genes to produce the hermaphrodite fate Loss of function mutations in tra 2 or tra 1 transform XX animals into males The transformation by tra 2 mutations can be suppressed by certain mutations in the tra 1 gene XX animals that are doubly mutant for a loss of function tra 2 mutation and a gain offunction tra 1 mutation develop as hermaphrodites As with the CYC7 suppressors described above the tra 1 suppressors bypass the requirement for another gene The difference is that tra 2 and tra 1 act in the same pathway whereas CYC1 and CYC7 act in parallel pathways How can you distinguish between bypass suppressors in the same or in parallel pathways Analysis of double lossof function mutants can sometimes be used to distinguish between the two In the case of parallel pathways the double mutants will result in a more severe phenotype In the cyc1 cyc2 double mutant for example no cytochrome c is produced whereas some cytochrome c is produced in either single loss of function mutant The tra 2 tra 1 double mutant by contrast has the identical phenotype to either single mutant Negative regulation A third example of bypass suppression is in a pathway containing a negative regulatory interaction In this case a mutation in a negative regulatory gene produces a phenotype because the gene downstream is activated inappropriately But a loss of function mutation in the downstream gene suppresses this phenotype It is worth noting that inactivating the downstream gene usually leads not only to suppression but to an additional and often opposite phenotype One good example of this type of suppression is in C elegans sexual development One of the genes that the transcription factor TRA 1 negatively regulates in XX animals is the cell death gene egl 1 which is required for programmed cell death in C elegans A sexually dimorphic cell in C elegans is the HSN motor neuron which innervates vulval muscles and stimulates egg laying by hermaphrodites Males don t lay eggs and hence don t need HSNs the HSNs undergo programmed cell death in males In XX animals lacking tra 1 function the HSNs adopt the male fate and die because the cell death gene egl 1 is expressed and activates the cell death pathway But eliminating egl 1 function in a tra 1 XX animal suppresses the cell death phenotype caused by the tra 1 mutation This suppression occurs only for the HSNs Cells other than the HSNs still adopt male fates in XX animals containing mutations in tra 1 and egl 1 because TRA 1 regulates other genes in these cells Interaction suppressors are compensating mutations in physically interacting components A mutation in one protein disrupts the interaction 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 this interaction is essential for viability Many proteins bind to actin to regulate actin assembly in cells In order to find proteins that regulate actin assembly in vivo suppressors of a temperature sensitive allele of the yeast actin gene ACT1 were isolated A diploid homozygous act1 act1 mutant was mutagenized and hence only dominant suppressors were isolated The investigators assumed that the original actin mutation impaired the interaction of an actin binding protein with actin They reasoned that isolation of dominant suppressors was more likely to yield mutations in genes that encode interacting proteins because such mutants would be mutations that restored interactions that were disrupted by the actin mutation Some suppressors were in the gene SAC6 which was later shown to encode the actin binding protein fimbrin When the sac6 mutations were separated from the act1 mutation it was found that the sac6 mutants were also inviable at higher temperatures Thus the two mutants act1 and sac6 were on their own very sick but the act1
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