section24_ak - MIT Department of Biology 7.014 Introductory...

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MIT Department of Biology 7.014 Introductory Biology, Spring 2005 Recitation Section 24 Answer Key May 11-12, 2005 Designing Yeast Assays for Human Disease A. Determining severity of disease phenotype We are again working with the human gene CBS and its yeast analog cys4. Recall that these genes each encode the protein cystathionine β -synthase that is responsible for converting homocysteine into cystathionine in the cellular pathway of creating cysteine. Human disease homocysteinurea results from CBS enzyme deficiency. Human cells deficient in CBS enzyme accumulate homocysteine—a product of the previous step in the pathway of making cysteine. This accumulation leads to a host of symptoms in humans, but not in yeast. In Section 22 we discussed why we expect wild type human CBS protein to complement CBS protein deficiency in yeast. 1. Briefly describe the procedure for expressing human CBS protein in cys4 - yeast. To express the human protein in the yeast cell, we can 1) design PCR primers to selectively amplify the mature mRNA for CBS protein (avoid introns and human promoter); 2) clone that human gene into a yeast vector under a yeast promoter; 3) transform a cys4 - strain of yeast with the library of vectors 4) select for the cells that picked up a vector (special tricks can be applied to make sure only the vectors with inserts are picked for analysis); 5) induce the yeast promoter to make the cell produce human protein. And, as a bonus, we can now 6) check to see whether the deficiency is complemented. We will now consider various disease alleles of homocysteinurea in the context of the same assay. Aside from scientific curiosity, there are clinical reasons for us to consider analyzing various disease alleles. 2. We do not expect all the disease alleles to behave similarly in this assay. Why not? Disease phenotype is caused by a number of different alleles of the CBS gene. We know that mutants with the same or similar phenotype may be a result of mutations in different parts of the CBS gene. Each of the mutant alleles will produce a protein that has different shape, and may behave differently depending on conditions. Because yeast system where the protein will be expressed is somewhat different from the human cells, we can not predict the behavior of each individual allele. When the wild type and various human disease alleles are put through this test, only WT human and V320A
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This note was uploaded on 05/02/2009 for the course BIOL 7.014 taught by Professor Walker during the Spring '05 term at MIT.

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section24_ak - MIT Department of Biology 7.014 Introductory...

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