Reporter Gene Transcription

Written by Super User. Posted in DNA

Learn about reporter gene transcription.

Learn about reporter gene transcription.

Reporter Gene Transcription

An important way to also measure transcription in vivo is to place a surrogate reporter gene under control of a specific promoter, and then measure the accumulation of the product of this reporter gene. If for example we are interested to probe the structure of a eukaryotic promoter. One way to do this is to make mutations in the DNA region that contains the promoter, then introduce the mutated DNA into cells and measure the effects of the mutations on promoter activity. We can use S1 mapping or primer extension analysis to do this measurement, but we can also substitute a reporter gene for the natural gene, and then assay the activity of the reporter gene product.

The reason why would conduct our investigation this way is because reporter genes have been carefully chosen to have products that are very convenient to assay-more convenient than S1 mapping or primer extension.

Some Bacterial Reporter Genes

lacZ: One of the most popular reporter genes, whose product, B-galactosidase can be measured using the substrate X-gal, which turns blue upon cleavage.

cat Reporter Gene: cat is a reporter gene who encodes the enzyme chloramphenicol acetyl transferase (CAT). Growth of most bacteria is inhibited by the antibiotic chloroamphenicol (CAM), which blocks a key step in protein synthesis. Some bacteria though have developed a means of evading this antibiotic by acetylating it and therefore blocking its activity. The enzyme that carries out this acetylation is CAT. But eukaryotes are not susceptible to this antibiotic, so they have no need for CAT. Therefore the background level of CAT activity in eukaryotic cells is zero. Which means that we can introduce a cat gene into these cells, under control of a eukaryotic promoter and any CAT activity we observe is due to the introduced gene. The way that we measure CAT activity in cells that have been transfected with the cat gene is by mixing an extract of our transfected cells with radioactive CAM and an acetyl donor (acetyl CoA). Then we use thin layer chromatography to separate CAM from its acetylated products. The greater concentrations of these products we see, the higher the CAT activity and therefore the higher the promoter activity.

Luciferase reporter gene: The luciferase reporter gene is found from firefly lanterns. The enzyme luciferase, mixed with ATP and luciferin, converts the luciferin to a chemoluminescent compound that emits light. This makes a convenient reporter because the light can be detected easily with x-ray film, or even a scintillation counter.

In the reporter gene experiments described above we are assuming that the amount of reporter gene product is a reasonable measure of transcription rate, and therefore of promoter activity. But the gene products come from a two-step process that includes translation as well as transcription. Usually we are justified in assuming that the translation rates do not vary from one construct to another, as long as we are manipulating only the promoter. That is because the promoter lies outside the coding region. For this reason changes in the promoter cannot affect the structure of the mRNA itself and therefore should not affect translation. However, we can deliberately make changes in the region of a gene that will be transcribed into mRNA, and then use our reporter to measure the effects of these changes in translation. Thus, depending on where the changes to a gene are made a reporter gene can detect alterations in either transcription or translation rates.

See the DNA Molecule in 3-Dimensions

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