Genetic Analysis: An Integrated Approach (2nd Edition)
2nd Edition
ISBN: 9780321948908
Author: Mark F. Sanders, John L. Bowman
Publisher: PEARSON
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Chapter 16, Problem 10P
In enhancer trapping experiments, a minimal promoter and a reporter gene are placed adjacent to the end of a transposon so that genomic enhancers adjacent to the insertion site can act to drive expression of the reporter gene. In a modification of this approach, a series of enhancers and a promoter can be placed at the end of a transposon so that transcription is activated from the transposon into adjacent genomic DNA. What types of mutations do you expect to be induced by such a transposon in a mutagenesis experiment?
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GR and PPAR are transcription factors that bind to GRE and PPARE sequences respectively and activate transcription of genes. A reporter cell line is created in which the the green fluoresecent protein (GFP) is controlled by a GRE sequence and the pink fluorescent protein mCherry is under control of a PPARE sequence. If the gene for GR is introduced into the reporter cell line, the cells produce a green color. Chimeric proteins are created in which the DNA Binding Domains (DBD) and Activation Domains (AD) of the transcription factors are introduced into various cell lines. Match the following cell-types with the fluorescent color(s) you would expect the cells to produce.
What is the difference between a promoter proximal element and a distal enhancer? What are the similarities?
Recombinant expression in prokaryotic systems has numerous advantages when compared to eukaryotic systems, one of which is the ability to produce the protein of interest at high levels. For this, it is essential to use strong promoters and genetically modified bacteria capable of overexpressing the exogenous gene. Therefore, mark the alternative that best represents the set of bacterial promoters/strains for protein overexpression. *
A)use of the T7 promoter, whose induction occurs by the addition of IPTG in the culture medium, and use of the Escherichia coli strain BL21DE3.
B)use of the T7 promoter, whose induction occurs by the addition of IPTG in the culture medium, and use of the Escherichia coli DH5 strain.
C)use of the lac promoter, whose induction occurs by the addition of IPTG in the culture medium, and use of the Escherichia coli DH5 strain.
D) use of the constitutive trp promoter, whose induction occurs by the addition of the amino acid Tryptophan in the medium, and use of…
Chapter 16 Solutions
Genetic Analysis: An Integrated Approach (2nd Edition)
Ch. 16 - 14.1 What are the advantages and disadvantages of...Ch. 16 - Prob. 2PCh. 16 - 3. Genetic maps and physical maps are both...Ch. 16 - 14.5 What are the advantages and disadvantages of...Ch. 16 - 14.6 You have cloned the mouse ortholog (see...Ch. 16 - 14.13 The CBF genes of Arabidopsis are induced by...Ch. 16 - 14.14 When the S. cerevisiae genome was sequenced,...Ch. 16 - 14.15 Translational fusions between a protein of...Ch. 16 - In enhancer trapping experiments, a minimal...Ch. 16 - 14.19 In Genetic Analysis, we designed a screen to...
Ch. 16 - How would you design a genetic screen to find...Ch. 16 - 14.21 The eyes of Drosophila develop from imaginal...Ch. 16 - 14.22 Given your knowledge of the genetic tools...Ch. 16 - Mutations in the CFTR gene result in cystic...Ch. 16 - Prob. 16PCh. 16 - 14.25 How would you conduct a screen to identify...Ch. 16 - In land plants, there is an alternation of...Ch. 16 - 14.27 The Drosophila evenskipped (eve) gene is...Ch. 16 - Prob. 20PCh. 16 - 14.29 As shown in Figure, mutations in the...
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- When Laybourne and Kadonaga studied the effects of histone proteins on eukaryotic transcription using an in vitro transcription assay explain why: a) they used two different DNA templates that contained different promoter structures. b) when they included both activator protein and histones, they always added the histone proteins before adding the activator to the transcription assay mixture. (Ctri) -arrow_forwardNegative supercoiling of DNA favors the transcription of genes because it facilitates unwinding. However, not all promoter sites are stimulated by negative supercoiling. The promoter site for topoisomerase II itself is a noteworthy exception. Negative supercoiling decreases the rate of transcription of this gene. Propose a possible mechanism for this effect and suggest a reason why it may occur.arrow_forwardennar region of gene X, which determines the length of the tail in mice, is mutated so that transcription factors bind it at a much higher affinity compared to the wild-type sequence. What is the most likely phenotypic outcome? Tail length will not change because the enhancer is a non-coding sequence Tail length will increased due to increased activity of the gene's promoter Tail length will decreased because any mutation will cause a loss-of-function of these regulatory regions Not just the tail will be enlarged because increased activity of the enhancer will impact many genesarrow_forward
- You have isolated two different mutants (reg1 and reg2) causing constitutive expression of the emu operon (emu1 emu2). One mutant contains a defect in a DNA-binding site, and the other has a loss-of-function defect in the gene encoding a protein that binds to the site. Is the DNA-binding protein a positive or negative regulator of gene expression? Explain. To determine which mutant has a defect in the site and which one has a mutation in the binding protein, you decide to do an analysis using F′ plasmids. Assuming you can assay levels of the Emu1 and Emu2 proteins, what results do you predict for the two strains (i and ii; see descriptions below) if reg2 encodes the regulatory protein and reg1 is the regulatory site? Explain. F′ (reg1− reg2+ emu1− emu2+)/reg1+ reg2+ emu1+ emu2− F′ (reg1+ reg2− emu1− emu2+)/reg1+ reg2+ emu1+ emu2−arrow_forwardAn electrophoretic mobility shift assay can be used to study the binding of proteins to a segment of DNA. In the results shown here, an EMSA was used to examine the requirements for the binding of RNA polymerase |l (from eukaryotic cells) to the promoter of a protein-encoding gene. The assembly of general transcription factors and RNA polymerase Il at the core promoter is described in Week 4. In this experiment, the segment of DNA containing a promoter sequence was 1100 bp in length. The fragment was mixed with various combinations of proteins and then subjected to an EMSA. Lane 1: No proteins added Lane 2: TFIID Lane 3: TFIIB Lane 4: RNA polymerase IIl Lane 5: TFIID + TFIIB Lane 6: TFIID + RNA 1 2 3 4 5 6. 7 polymerase II Lane 7: TFIID + TFIIB + RNA polymerase Il 1100 bp Explain the results.arrow_forwardAn enhancer is surrounded by four genes (A, B, C, and D), as shown in the accompanying diagram. An insulator lies between gene C and gene D. On the basis of the positions of the genes, the enhancer, and the insulator, the transcription of which genes is most likely to be stimulated by the enhancer? Explain your reasoning.arrow_forward
- You have isolated different mutants (reg1 and reg2) causing constitutive expression of the emu operon (which has genes emu1 and emu2). One mutant contains a defect in a DNA-binding site, and the other has a loss-of-function defect in the gene encoding a protein that binds to the site. Is the DNA-binding protein a positive or negative regulator of gene expression?arrow_forwardExplain how a transcriptional fusion to a reporter gene, combined with site directed mutagenesis, can indicate key nucleotides in a regulatory region. Name one in vitro technique that can be used to further confirm the significance of these regions.arrow_forwardInversion of an enhancer region does not affect gene expression, but inversion of the promoter region completely abolishes gene expression. Explain why this is true.arrow_forward
- You are studying how the expression of Gene F is regulated. Initially, you identified three potential regulatory elements in the regulatory region upstream of Gene F's transcription start site. To study these potential regulatory elements, you created three different mutants with a different potential regulatory element mutated in each. The data below shows qPCR data for expression of Gene F in Wild-type (Control) samples, each of the three different mutants (Mutant 1-3), and a negative control (No DNA Control). Were you able to identify a regulatory element that binds a transcriptional activator? If so, explain how you determined that and which mutant it is. Make sure to reference specific data from the image and your knowledge of the molecular biology involved in regulating transcription. The dotted line represents our measurement threshold. Fluorescence N Wild-Type Control Mutant 1 Mutant 2 Mutant 3 No DNA Control 10 10 20 30 Cyclearrow_forwardBacterial DNA containing an operon encoding three enzymes is introduced into chromosomal DNA in yeast (a eukaryote) in such a way that it is properly flanked by a promoter and a transcriptional terminator. The bacterial DNA is transcribed and the RNA correctly processed, but only the protein nearest the promoter is produced. Can you suggest why?arrow_forwardIn the laboratory, you want to study protein that is normally toxic to E. coli cells. You wish to grow this protein in E. coli and purify it from E. coli. Your advisor suggests cloning the gene into an expression vector that uses the araBAD promoter. Explain why is it ideal to use the araBAD promoter for expression of your gene of interest in E. coli cells?arrow_forward
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