E the exons and blast it against the est database and

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Unformatted text preview: ast it against the EST database and if there are any hits then you can check what tissue/developmental time point the ESTs were cloned from. (b) You decide to recreate in mice the mutation found in the affected individuals. Based on the human disease phenotype your supervisor thinks that Epidermo-1 may be involved 7.06 Spring 2004 PS 1 key 8 of 11 in forming cell-cell junctions and therefore may be essential during development of the mouse. How would you modify your gene targeting strategy so as to ensure the targeted mice were not embryonic lethal? LoxP sites are shown in yellow, the neomycin resistance gene is shown in green. 1 2 2 3 NeoR 4 3 Using the loxP sites allows for a conditional knockout of exon 3. Once the targeted mice have been generated they need to be crossed to a mouse strain that expresses Cre in the skin. (c) The mutant mice you generate develop a skin blistering disease. To try and investigate the function of Epidermo-1 you take skin sections from both wild type and mutant mice and using an antibody against the N-terminus you carry out some immunostaining. In wild-type skin Epidermo-1 is found to co-localize with desmocollin and desmoglein, two proteins found in desmosomes. Since a portion of exon 3 is composed of a stretch of 22 hydrophobic amino acids that can form an a -helix, where might you expect to find the mutant Epidermo-1 protein? Why? You might expect to find the mutant protein in the cytoplasm since it is missing the part of the protein that is likely to be the transmembrane portion. The interior of the plasma membrane is hydrophobic and therefore amino acids that are in contact with the fatty acyl chains of the bilayer need to have hydrophobic side-chains. Stretches of 20-25 hydrophobic amino acids can form an a -helix that traverses the plasma membrane. This is the principle secondary structure that the membranespanning portion of a protein takes, the other being the b-barrel – a single b-sheet is not sufficient to satisfy all of the H-bonds in the polypeptide backbone. Both structures maximize H-bo...
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