Once a gene has been identified as being inducible under certain inducing conditions,in this case in the presence of galactose,we can begin to dissect the regulatory mechanism by isolating mutants;i.e.,mutants that constitutively express the GAL genes even in the absence of galactose,and mutants that have lost the ability to induce the GAL genes in the presence of galactose.If we were studying galactose regulation today we would probably use a lacZ reporter system as we discussed in the last lecture.However, when the Gal regulatory system was fist genetically dissected,it was done by actually measuring the induction of Gall encoded galactokiase activity,so this is how we will discuss the genetic dissection of the system. Mutagenized GAL1::Tn7lacZ fusion strain grown on: Another approach is to simply measure galactokinase GLYCEROL activity in the presence or absence of Galactose Gal regulatory mutants galactokinase activity -galactose galactose Interpretation Gal+ GLYCEROL GALACTOSE X-Gal +X-Gal Gal1- Gall-inactivates galactokinase Constitutive Gal4- Gal4-is uninducible Gal80- Gal80-is constitutive Uninducible Gal81- + Gal81-is constitutive What we know is that Gal4 mutants are uninducible and that Gal80 and Gal81 mutants constitutively express the Gall galactokinase gene,along with the other Gal genes.Let's analyze each mutant in turn: Gal4 mutant:It was first established that,like Gal1,the Gal4-mutant phenotype is recessive,because heterozygous diploids generated by mating Gal4 to wild type have normal regulation.It was then established that the Analysis of Gal4 mutation in the Gal4-strain lies in a new gene,and not simply in the GAL1 Gal4-is uninducible galactokinase gene;Gall-mutants Gal4+Gal4-is regulated: don't express galactokinase activity in therefore Gal4-is recessive the presence of galactose,just as was MATa Gal1-Gal4+x MATa Gal1+Gal4- seen for the Gal4 mutant.That 1 4 Gal1-and Gal4 mutants have Type1 Type 2 Type 3 mutations in different genes was uninducible uninducible uninducible shown by complementation analysis, uninducible uninducible uninducible (diploids from mating Mata Gal4 uninducible uninducible regulated (wt) uninducible regulated (wt) regulated(wt) with Mata Gal1-behave like wild type)and the fact that the GAL4 andOnce a gene has been identified as being inducible under certain inducing conditions, in this case in the presence of galactose, we can begin to dissect the regulatory mechanism by isolating mutants; i.e., mutants that constitutively express the GAL genes even in the absence of galactose, and mutants that have lost the ability to induce the GAL genes in the presence of galactose. If we were studying galactose regulation today we would probably use a lacZ reporter system as we discussed in the last lecture. However, when the Gal regulatory system was fist genetically dissected, it was done by actually measuring the induction of Gal1 encoded galactokiase activity, so this is how we will discuss the genetic dissection of the system. Another approach is to simply measure galactokinase activity in the presence or absence of Galactose Another approach is to simply measure galactokinase activity in the presence or absence of Galactose GAL1::Tn7lacZ fusion strain grown on: GLYCEROL GLYCEROL + X-Gal GALACTOSE + X-Gal Constitutive Uninducible GAL1::Tn7lacZ fusion strain grown on: GLYCEROL GLYCEROL + X-Gal GALACTOSE + X-Gal GAL1::Tn7lacZ fusion strain grown on: GLYCEROL GLYCEROL + X-Gal GALACTOSE + X-Gal Constitutive Uninducible Mutagenized What we know is that Gal4 mutants are uninducible and that Gal80 and Gal81 mutants constitutively express the Gal1 galactokinase gene, along with the other Gal genes. Let’s analyze each mutant in turn: Gal4 mutant: It was first established that, like Gal1- , the Gal4- mutant phenotype is recessive, because heterozygous diploids generated by mating Gal4- to wild type have normal regulation. It was then established that the mutation in the Gal4- strain lies in a new gene, and not simply in the GAL1 galactokinase gene; Gal1- mutants don’t express galactokinase activity in the presence of galactose, just as was seen for the Gal4- mutant. That Gal1- and Gal4- mutants have mutations in different genes was shown by complementation analysis, (diploids from mating Matα Gal4- with Mata Gal1- behave like wild type) and the fact that the GAL4 and