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Lecture 20

Lecture 20: "Functional Genomics & Systems Biology II"

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Biology 2581B
Jim Karagiannis

Genetics Lecture No. 20: Functional Genomics & Systems Biology II th Monday March 25 , 2013 LECTURE 19 (CONT’D) Defining The Interactome (Protein-Protein): -To be able to transport proteins in and out of the nucleus, you need a specific structure. Over 60 proteins are required to come together and make a functional nuclear pore. This an example of why protein-protein interactions are so important as cellular physiology is often controlled by large networks of interacting proteins that come together at precise locations within the cell and at precise times during development or the cell cycle LECTURE 20 Defining The Interactome (Yeast Two Hybrid Assay): -The traditional way of defining the interactions between the molecular machines (proteins) is by performing a Yeast Two Hybrid Assay (an assay done within yeast cells for testing the interaction between two proteins of interest, X and Y). The creation of protein fusions is necessary where you fuse one protein (with the DNA-binding domain) to the promoter and have a separate polypeptide (with an activation domain) fuse to that bound protein. If protein X and protein Y do not interact, the activation domain is never going to come into contact with your binding domain and no activation of the reporter gene will result. If they do in fact interact, the activation and binding domain will come into close proximity, allowing for the activation domain to activate transcription of the reporter. Note that even though this assay is performed yeast, you can use proteins from any organism. -In this assay, you create a haploid budding yeast strain that has a plasmid expressing a gene that codes for your fusion. Your first protein of interest is often referred to as the bait (B), the one that is fused to the DNA-binding domain (DBD). A second haploid strain has a plasmid expressing the second fusion (second protein of interest), with the activation domain (AD) and open reading frame (ORF). These two haploid strains are mated to produce a diploid strain expressing both proteins of interest (fusions). The activation of the HIS3 reporter gene (involved in histidine biosynthesis) for transcription tells you if the two proteins interact with each other (i.e. if proteins fail to interact, the yeast cells will not be able to survive in a media lacking histidine). Using The Yeast Two Hybrid Assay On A Genome-Wide Scale: -Here we want to test protein X and its interaction with every other protein in the genome using a macroarray and some robotics. Create a set of strains equivalent to the number of genes in the genome (about 6,000 strains), each expressing a fusion of the GAL4-AD to one of the genes in the genome. On a plate, each circle represents a yeast colony where the GAL4-AD is fusing with a specific gene (one plate can hold 96 colonies or 96 different fusions). An actual macroarray plate can hold 1,536 colonies or more, demonstrating how easy it is to represent the entire genome (uses about 17 of these plates). -In another macroarray, your bait or query protein will be expressed by all the yeast colonies on the plate (each colony is expressing the identical fusion between the DBD and the query protein). The formation of diploids expressing both the AD and BD fusion can be done manually, but more often it is performed through robotics. A replica pinning tool (steel block with metal pins) stamps down onto your macroarray (where a few cells will get stuck to the metal pins) and stamp down onto the other array (mixing the cells from both arrays, where they can fuse and form a diploid). For the resultant diploid cells will grow on the +HIS plate, thus the robot is used to transfer some of those diploid cells onto growth media lacking histidine (-HIS), where only diploids expressing interacting fusion proteins activate the reporter gene and are able to grow. Using this technology, you can define all the interactors of your query strain to every single gene in the genome. Disadvantages Of The Yeast Two-Hybrid System: -The Yeast Two-Hybrid System is slightly old-fashioned since the assay is performed only in yeast cells, which is not optimal if we want to study the protein interactions in humans or other organisms. Another disadvantage is that the presence of AD or DBD (artificial) could quite possibly interfere with proper protein folding (incorrect folding may interfere with the ability of such proteins to interact). Defining The Interactome (Affinity Capture Coupled To Mass Spectrometry): -A more modern technique of achieving the same ends as the Yeast Two-Hybrid System is known as affinity capture coupled with mass spectrometry. Affinity capture is where an antibody is used to immunoprecipitate a protein of interest from any cellular extract under natural condi
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