MCAT Content / Recombinant Dna And Biotechnology / Determining Gene Function

Determining gene function

Topic: Recombinant Dna And Biotechnology

The sequencing of genomes has discovered many different genes and there are methods used to establish what the gene’s function is.

Evolutionary Comparisons

Genes that are discovered can be compared to sequences of other organisms. As an example, if a human gene that has an unknown function has most of its sequence in common with a fission yeast protein phosphatase a researcher will test if the unknown gene codes for the phosphatase.

Protein Domains

Protein domains are typically between 25 and 500 amino acids in length and contribute to protein function. They are distinct structural units of protein. Two examples of protein domains are zinc fingers and pleckstrin homology domains. Zinc fingers are small protein domains commonly found in transcription factors. Pleckstrin homology domains function in lipid binding, which targets proteins to cellular compartments. Pleckstrin homology domains occur in a range of proteins involved in signalling pathways.

Protein Interactions

Since proteins bind to certain proteins, their gene function can be determined by studying certain protein pathways. Immunoprecipitations are used to find protein binding partners. In immunoprecipitations, cell lysates are collected and incubated with an antibody of a specific protein of interest. As a result, complex forms and there is a microscopic bead added. The microscopic bead has an antibody binding protein linked to it. To be analyzed, the solution is centrifuged since this can pull the bead out of the solution allowing it to collect at the bottom. The complexes are then washed and purified with a lysate solution. The precipitated proteins are then analyzed by western blot or mass spectrometry. This data can be collated in network maps. These maps are used to demonstrate how the proteins of the gene are working together in the cell.

Cellular Expression

The location of a cell can help identify its proteins’ unknown function. A reporter system can be used to tag the gene of a certain protein to see where in the cell it is expressed. As a possible method, the gene can be cloned in an expression vector and linked to a fluorescent molecule such as GFP. Then the cellular location can be determined using a fluorescent microscope. This will determine if the gene is expressed in the plasma membrane, cytoplasm, or nucleus.


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Key Points

• Evolutionary comparisons, protein domains, protein interactions, and cellular expression are all used to help determine the function of a specific gene

• The evolutionary comparison uses the sequences of other organisms’ genomes

• Protein domains help determine gene function by comparing that specific gene’s domain to discovered ones and comparing function

• Protein interactions help determine gene function by gene network maps

• Cellular expression helps determine gene function by identifying a specific gene’s location thereby determining the protein’s function in that specific location

Key Terms

western blot: an adaptation of the Southern blot procedure, used to identify specific amino-acid sequences in proteins.

mass spectrometry: an apparatus for separating isotopes, molecules, and molecular fragments according to mass.

gene network: describes biological interactions among genes and provides a systematic understanding of cellular signalling and regulatory processes

phosphatase: an enzyme that catalyzes the hydrolysis of organic phosphates

zinc fingers: small protein domains commonly found in transcription factors

pleckstrin homology domains: function in lipid binding, which targets proteins to cellular compartments

immunoprecipitation: the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein.

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