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FIGURE 9-24 Use of comparative genomics to identify functionally related genes. One use of comparative genomics is to prepare phylo-genetic profiles in order to identify genes that always appear together in a genome. This example shows a comparison of genes from four organisms, but in practice, computer searches can look at dozens of species. The designations P1, P2, and so forth refer to proteins encoded by each species. This technique does not require homologous proteins. In this example, because proteins P3 and P6 always appear together in a genome they may be functionally related. Further testing would be needed to confirm this inference.

on the column. The connection between the protein and the tag is cleaved with a specific protease, and the protein complexes are eluted from the column and analyzed. Researchers can use these methods to define complex networks of interactions within a cell.

A variety of useful protein tags are available. A common one is a histidine tag, often just a string of six His residues. A poly-His sequence binds quite tightly to metals such as nickel. If a protein is cloned so that its sequence is contiguous with a His tag, it will have the extra His residues at its carboxyl terminus. The protein can then be purified by chromatography on columns with immobilized nickel. These procedures are convenient but require caution, because the additional amino acid residues in an epitope or His tag can affect protein activity.

Yeast Two-Hybrid Analysis A sophisticated genetic approach to defining protein-protein interactions is based on the properties of the Gal4 protein (Gal4p), which activates transcription of certain genes in yeast (see Fig. 28-28). Gal4p has two domains, one that binds to a specific DNA sequence and another that activates the RNA polymerase that synthesizes mRNA from an adjacent reporter gene. The domains are stable when separated, but activation of the RNA polymerase requires interaction with the activation domain, which in turn requires positioning by the DNA-binding domain. Hence, the domains must be brought together to function correctly (Fig. 9-25a).

Gal4p DNA-binding domain

Gal4p DNA-binding domain

Reporter gene

Yeast strain 1 with Gal4p-binding domain fusions

Survivors form colonies.

FIGURE 9-25 The yeast two-hybrid system. (a) In this system for detecting protein-protein interactions, the aim is to bring together the DNA-binding domain and the activation domain of the yeast Gal4 protein through the interaction of two proteins, X and Y, to which each domain is fused. This interaction is accompanied by the expression of a reporter gene. (b) The two fusions are created in separate yeast strains, which are then mated. The mated mixture is plated on a medium on which the yeast cannot survive unless the reporter gene is expressed. Thus, all surviving colonies have interacting protein fusion pairs. Sequencing of the fusion proteins in the survivors reveals which proteins are interacting. ^ Yeast Two-Hybrid Systems

Yeast strain 2 with Gal4p-activation domain fusions

Mate to produce diploid cells.

Plate on medium requiring interaction of the binding and activation domains for cell survival.

Survivors form colonies.

Sequence fusion proteins to identify which proteins are interacting.

FIGURE 9-25 The yeast two-hybrid system. (a) In this system for detecting protein-protein interactions, the aim is to bring together the DNA-binding domain and the activation domain of the yeast Gal4 protein through the interaction of two proteins, X and Y, to which each domain is fused. This interaction is accompanied by the expression of a reporter gene. (b) The two fusions are created in separate yeast strains, which are then mated. The mated mixture is plated on a medium on which the yeast cannot survive unless the reporter gene is expressed. Thus, all surviving colonies have interacting protein fusion pairs. Sequencing of the fusion proteins in the survivors reveals which proteins are interacting. ^ Yeast Two-Hybrid Systems l

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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