lectures 10_20-10_24

lectures 10_20-10_24 - Announcements Exam on Monday 10/27...

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1 Announcements Exam on Monday 10/27 covers: • Lecture slides (except Chapter 11 slides) • Chapter 8-10 • Chapter 21(through DNA sequencing, p. 758) • Chapter 22 ( S. cerevisiae : p. 795-98, mouse: 812-16 Office hours on Friday from 2:30-3:30 only! Knocking-Out a Gene in Yeast by Homologous Recombination A DNA construct with a selectable marker flanked by homology to the gene of interest is transformed into yeast. Transformants that have inserted the DNA into the genome can be identified by the function of the selectable marker. Homologous recombination by both ends of the transformed DNA results in a gene replacement (two crossovers occur). Result of epitope tagging a protein at the C-terminus • The attached epitope forms its own “domain” • Tagging requires construction of a gene fusion at the DNA level to produce the epitope-tagged protein Epitope C-terminus
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2 Tagging a Gene (Epitope tagging) • Insertion of additional DNA coding sequences at the beginning or end of an Open Reading Frame (ORF) generates a gene fusion that will give rise to a protein fusion • Epitopes are short segments (~7-10) of amino acids recognized by an antibody • Commonly used sequences called “epitope tags” are fused to genes to allow analysis of the proteins by available antibodies that recognize the epitope N-terminal fusion C-terminal fusion Open Reading Frame Tagging a Gene (Epitope tagging) • An in vitro -produced DNA construct with the epitope tag fused, in the correct reading frame , to the gene of interest, and with upstream and downstream homology flanking the site of insertion, is transformed or transfected in vivo. • DNA insertion occurs through homologous recombination • A selectable marker (not shown) is usually required. Gene X Tag Gene X+Tag Homologous recombination (two crossovers) Green Fluorescent Protein • Isolated from jellyfish • Naturally fluoresces due to a special structure in the protein (a cyclization of three amino acids forms the fluorophore) • Useful as an in vivo (or in vitro ) protein tag • 2008 Nobel Prize in Chemistry for its discovery (), application (Chalfie), and enhancing its use (Tsien)
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3 Result of GFP-tagging a protein • GFP forms its own domain and fluoresces • The intracellular location of the protein may be followed C-terminus Conditional Mutations Are Used To Analyze the Function of Essential Genes Essential genes are genes whose deletion (null mutation) causes lethality of the organism. To analyze the specific function of an essential gene in a living cell, a conditional
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lectures 10_20-10_24 - Announcements Exam on Monday 10/27...

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