Consider a hypothetical gene encoding an enzyme called LARDA

Consider a hypothetical gene encoding an enzyme called LARDASE that is involved in the degradation of fats in mice. This gene is expressed specifically in liver and muscle, and it is induced by glucocorticoid hormone (steroid hormone that bind to the glucocorticoid receptor (GR), and plays a role in the regulation of the metabolism of glucose). To dissect the organization of the LARDASE promoter region, you undertake a mutational analysis. You use lacZ as a \"reporter gene\" to monitor promoter activity: the first 500 bp of DNA upstream of the LARDASE transcription start site (i.e. -500 to +1) is fused to the lacZ gene, as shown below. You prepare various mutant promoters containing increasing and decreasing amounts of upstream sequence, as shown. You then introduce the mutant DNAs into cultured mouse muscle or liver cells, incubate the cells in the presence or absence of glucocorticoid hormone, and measure ft-galactosida.se activity after several hours. The results are shown below. Why must a reporter gene be used to monitor promoter activity in this type of experiment? That is, why not measure lardase expression itself? Do your best to diagram the locations of all of the m-acting sites detected in these experiments. Be sure to explicitly specify the role of each site in regulating this gene in each tissue type. Based on the information from the above experiment, describe the organization of regulator)\' sequences between -70 and -140 and how this organization explains the data. Don\'t try to memorize structures, think about the data.

Solution

a) Use of reporter gene: The reporter gene helps us to determine and quantitate the expression of any gene of interest. The addition of LacZ in this case, helps easy determination of promoter activity. This helps to rule out any unwanted changes arising in expression of LARDASEe due to mutation in promoter region.

b) The deletion of region between -100 to – 70 removes the CAAT box situates around -60 to -100 region. Removal of this region, inhibits the binding on core binding factor which in turn reduce binding of RNA pol II.

Deletion till 250 removes CREB elements. CREB (cAMP response element-binding protein) is a cellular transcription factor. It binds to certain DNA sequences called cAMP response elements (CRE), thereby increasing or decreasing the transcription of the downstream genes.