706_S2006_PS1key - 7.06 Problem Set One 2006 ANSWER KEY...

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1 7.06 Problem Set One, 2006 – ANSWER KEY 1. (Note: This problem is based on the background knowledge in molecular biology, genetics, and biochemistry that you are presumed to have as a student coming into 7.06.) A recent study done in Schizosaccharomyces pombe (fission yeast) showed that the CHZ1 gene is involved in metabolizing the tasty bright orange cheese-flavored powder on Cheetos as an alternative carbon source. A chz1 knockout in S. pombe is inviable on medium in which Cheeto powder is the only carbon source. Intrigued by this novel metabolic pathway, you perform a BLAST search and find a CHZ1 homolog in your organism of choice, Saccharomyces cerevisiae (budding yeast). Your first goal is to knock out CHZ1 in budding yeast and examine the phenotype. a) Describe how you would make a knockout of CHZ1 in S. cerevisiae. The easiest way to knockout CHZ1 in yeast would be to use homologous recombination to disrupt the endogenous gene with a selectable marker. To do so, you would need to transform haploid yeast with a DNA fragment containing the selectable marker gene flanked by short sequences that are homologous to your target gene. Homologous recombinants carrying the deleted chz1 would then be identified by plating on selective media. (This is assuming that CHZ1 is not essential; if it is, you won’t be able to isolate the knockout in haploid yeast. An alternative method would be to make the knockout in diploids to get heterozygous diploids.) b) You plate your chz1 mutant strain of Saccharomyces cerevisiae on medium in which Cheeto powder is the only carbon source, but the strain manages to grow and there is no obvious phenotype. Propose possible explanations for your finding. Possible Explanations: 1) There are other genes that are redundant in function with CHZ1. 2) Even though there may be sequence homology between S. pombe CHZ1 and S. cerevisiae CHZ1 , function is not conserved. 3) CHZ1 isn’t really knocked out in your strain and is still functional. (Hey, it happens.) Did you confirm the deletion by PCR, immunoblotting for the presence/absence of Chz1 protein, etc.? Is the CHZ1 gene single-copy, or are there multiple copies of CHZ1 in the genome that didn’t get knocked out? Maybe S. cerevisiae has multiple homologs of S. pombe CHZ1 . c) You decide to search for other genes involved in Cheeto powder metabolism. Luckily, because you work in S. cerevisiae , you have a very important tool available to you – the S. cerevisiae deletion collection. This is a collection of thousands of yeast strains, each of which lacks a single non-essential gene from the genome. Using the S. cerevisiae deletion collection as a starting point, describe how you would perform the genetic screen of interest to you. A simple screen to look for genes involved in Cheeto powder metabolism would be to plate
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This note was uploaded on 01/23/2012 for the course BIOLOGY lsm1301 taught by Professor Seow during the Spring '11 term at National University of Singapore.

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706_S2006_PS1key - 7.06 Problem Set One 2006 ANSWER KEY...

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