2. A human gene X has been identified which, when mutated, causes a genetic illness. The sequence of gene X has been determined, but the function of the gene is not known. Sequence analysis reveals that the gene has a high degree of homology with a yeast gene of known function. You would like to determine if human gene X has the same function as the yeast gene. You have obtained a yeast mutant which has a deletion mutation in this gene. The phenotype of the yeast mutant is that it grows normally at 30°C but cannot grow at the elevated temperature of 37°C. You hypothesize that if the human gene has the same function as the yeast gene, then expressing the human gene in the yeast mutant will rescue the mutant phenotype, enabling it to grow at 37°C. Address the following questions regarding the steps you will take to test the hypothesis:
a. You have a source of total human cDNA. How will you clone the specific cDNA for gene X? (Do not just name the technique that you will use, but list the specific steps).
b. You would like to clone the cDNA of X into a plasmid that can be amplified in bacteria and from which reasonable yields of protein can be obtained by expression in yeast. List all the features that this plasmid must have.
2. A human gene X has been identified which, when mutated, causes a genetic illness. The sequence of gene X has been determined, but the function of the gene is not known. Sequence analysis reveals that the gene has a high degree of homology with a yeast gene of known function. You would like to determine if human gene X has the same function as the yeast gene. You have obtained a yeast mutant which has a deletion mutation in this gene. The phenotype of the yeast mutant is that it grows normally at 30°C but cannot grow at the elevated temperature of 37°C. You hypothesize that if the human gene has the same function as the yeast gene, then expressing the human gene in the yeast mutant will rescue the mutant phenotype, enabling it to grow at 37°C. Address the following questions regarding the steps you will take to test the hypothesis:
a. You have a source of total human cDNA. How will you clone the specific cDNA for gene X? (Do not just name the technique that you will use, but list the specific steps).
b. You would like to clone the cDNA of X into a plasmid that can be amplified in bacteria and from which reasonable yields of protein can be obtained by expression in yeast. List all the features that this plasmid must have.
