A biotech company sells a "reporter assay kit" for researchers to easily find small molecules that can activate the Glucocorticoid Receptor (GR), a type of hormone receptor. In such an assay, a synthetic transcriptional activator is expressed that has a Gal4 DNA binding domain fused to a GR activation domain/ligand binding domain as shown in the figure. This activator is designed to bind to a synthetic piece of DNA, where it is upstream of the gene encoding for luciferase, an enzyme that produces bioluminescence in organisms such as fireflies. In other words, if the luciferase gene is expressed, the cell will emit light (aka "glow"). (b) GR GR Gal4 Gal4 Luciferase gene (a) What DNA sequence should be in place upstream of the luciferase gene (as indicated in dark blue in the figure) to ensure that this assay works? If one adds galactose to the cell, will you observe light emission from the cell? (c) If one adds a small molecule that activates GR (such as cortisol) to the cell, will you observe light emission from the cell? (d) GR is a type I hormone receptor. When cortisol, a known ligand for GR, is added to the cell, where in the cell do you expect to see free, unbound cortisol?

A biotech company sells a "reporter assay kit" for researchers to easily find small molecules that can activate the Glucocorticoid Receptor (GR), a type of hormone receptor. In such an assay, a synthetic transcriptional activator is expressed that has a Gal4 DNA binding domain fused to a GR activation domain/ligand binding domain as shown in the figure. This activator is designed to bind to a synthetic piece of DNA, where it is upstream of the gene encoding for luciferase, an enzyme that produces bioluminescence in organisms such as fireflies. In other words, if the luciferase gene is expressed, the cell will emit light (aka "glow"). (b) GR GR Gal4 Gal4 Luciferase gene (a) What DNA sequence should be in place upstream of the luciferase gene (as indicated in dark blue in the figure) to ensure that this assay works? If one adds galactose to the cell, will you observe light emission from the cell? (c) If one adds a small molecule that activates GR (such as cortisol) to the cell, will you observe light emission from the cell? (d) GR is a type I hormone receptor. When cortisol, a known ligand for GR, is added to the cell, where in the cell do you expect to see free, unbound cortisol?

Biology (MindTap Course List)

11th Edition

ISBN:9781337392938

Author:Eldra Solomon, Charles Martin, Diana W. Martin, Linda R. Berg

Publisher:Eldra Solomon, Charles Martin, Diana W. Martin, Linda R. Berg

Chapter14: Gene Regulation

Section: Chapter Questions

Problem 8TYU: Through alternative splicing, eukaryotes (a) reinforce gene inactivation (b) prevent transcription...

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The transcription of a gene called YFG (your favoritegene) is activated when three transcription factors (TFA,TFB, TFC) interact to recruit the co-activator CRX. TFA,TFB, TFC, and CRX and their respective binding sitesconstitute an enhanceosome located 10 kb from the transcription start site. Draw a diagram showing how youthink the enhanceosome functions to recruit RNA polymerase to the promoter of YFG.
The codon change (Gly-12 to Val-12) in human rasH that convertsit to oncogenic rasH has been associated with many types ofcancers. For this reason, researchers would like to develop drugs toinhibit oncogenic rasH. Based on your understanding of the Rasprotein, what types of drugs might you develop? In other words,what would be the structure of the drugs, and how would theyinhibit Ras protein? How would you test the efficacy of the drugs?What might be some side effects?
Based on its sequence similarity to Apobec1,which deaminates Cs to Us in RNA, activation-induceddeaminase (AID) was originally proposed to work on RNA.But definitive experiments in E. coli demonstrated thatAID deaminates Cs to Us in DNA. The authors of the paperexpressed AID in bacteria and followed mutations in aselectable gene. They found that AID expression increasedmutations about fivefold above the background level inthe absence of AID expression. More importantly, theyfound that 80% of the induced mutations were G→A orC→T. Does this fit with your expectation if AID-inducedmutations arose by deamination of C to U in the DNA?
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. One mechanism by which antisense RNAs act as negative regulators of gene expression is by base pairingwith the ribosome binding site on the sense mRNA toblock translation. In a second, alternative mechanism,the act of transcribing an antisense RNA can somehow prevent RNA polymerase from recognizing thesense promoter for the same gene. Design an experimental approach that would enable you to distinguishbetween these two modes of action at a specific gene.(Hint: What would be the outcome in each case ifhigh levels of the antisense RNA were transcribedfrom a gene on a plasmid?)
Wilms tumor 1, or nephroblastoma, is caused by mutations in the WT1 gene, which encodes a transcription factor. You have identified a novel variant in WT1: Arg422Pro. You have control cells and cells that have been engineered to carry the homozygous WT1 p.Arg422Pro mutation. You want to assess effects of this mutation on a variety of endpoints. For each endpoint listed below, choose the one technique is best suited to answer the question. Choose from: array CGH, qRT-PCR, qPCR, RNA-seq, FISH, in situ hybridization, western blot, immunostaining, WT1 ChIP-seq, WT1 ChIP-PCR, ATAC-seq, 3C Endpoint Technique? WT1 protein amount (quantitative) Western blot WT1 protein binding to all enhancers, genome-wide Chip-seq WT1 mRNA amount (quantitative) WT1 protein subcellular localization Quantitative assessment of all mRNAs in these cells (genome-wide) RNAseq Chromatin interactions between a specific WT1 chromatin binding site (identified above)…
. An interesting mutation in lacI results in repressorswith 110-fold increased binding to both operator andnonoperator DNA. These repressors display a “reverse”induction curve, allowing β-galactosidase synthesis inthe absence of an inducer (IPTG) but partly repressingβ-galactosidase expression in the presence of IPTG. Howcan you explain this? (Note that, when IPTG binds a repressor, it does not completely destroy operator affinity,but rather it reduces affinity 110-fold. Additionally, ascells divide and new operators are generated by thesynthesis of daughter strands, the repressor must findthe new operators by searching along the DNA, rapidlybinding to nonoperator sequences and dissociating fromthem.)
Enhancers can influence the transcription of genes far away on the same chromosome. How are the effects of enhancers restricted so that they do not exert inappropriate transcriptional activation of non-target genes?
The binding of a small effector molecule, protein-protein interactions, and covalent modifications are three common ways to modulate the activities of transcription factors. Which of these three mechanisms are used by steroid receptors and by the CREB protein?
Chromatin Immunoprecipitation (ChIP) experiments enable researchers to measure the levels of transcription factors, coactivators/corepressors and chromatin remodeling complexes on a specific gene in cells. In the ChIP experiment below, the recruitment of the transcription factor NfkB, Histone Deacetylase Complex (HDAC3) and p300/CBP complex to the Interleukin 12 (IL12) gene at various times (0, 30, 60 and 120 min) after treating cells with LPS are measured. Does LPS stimulate or inhibit transcription of the IL12 gene based on the recruitment of factors to the IL12 gene. Justify your answer by explaining the state of the gene before LPS and after LPS treatment (limit 5-6 sentences).
RNAP can access the lacz promoter irrespective of whether CAP-CAMP is binding the CAP O True O False
Proteins that are retained in the ER have a KDEL sequence at their C-terminus. To test the idea that KDEL is both necessary and sufficient for retention in the ER, two experiments were performed. First, it was deleted from protein disulfide isomerase (PDI), and this form of PDI got secreted. Second, it was attached to a peptide growth hormone, which is normally secreted from the cell. This modified peptide still got secreted. Which interpretation does this support? The data do not allow for an interpretation, additional experiments are needed. KDEL is sufficient but not necessary for ER retention. KDEL is both necessary and sufficient for ER retention. KDEL is necessary but not sufficient for ER retention.