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Chapter 12: Problem 14

In your experiment in problem \(12,\) you discover a gene that is strongly expressed in anaerobically grown yeast but turned off in acrobically grown yeast. You name this gene nox (for "no oxygen"). You have the "bright idea" that you can engineer a yeast strain that senses \(\mathrm{O}_{2}\) levels if you can isolate the nox promoter. Describe how you might make a reporter gene construct using the nox promoter and how the yeast strain bearing this reporter gene construct might be an effective oxygen sensor.

Short Answer

Expert verified
To create a reporter gene construct using the nox promoter, the nox promoter is cloned in front of a reporter gene on a plasmid. This construct is inserted into yeast cells. When the yeast is grown anaerobically, the nox promoter is activated, leading to the expression of the reporter gene that produces a measurable signal, thus acting as an oxygen sensor. In the presence of oxygen, the nox promoter represses the reporter gene, and no signal is generated.

Step by step solution

01

Understand the basic concept

Understand that a promoter is a DNA sequence where the transcription of a gene begins. The nox gene is expressed under anaerobic conditions, so the nox promoter should be activated under these conditions. A reporter gene is a gene that is not present in the organism under study, but can be measured easily and accurately. It is tagged to the gene of interest so that the activity of the gene of interest can be studied. The reporter gene in this construct will be under the control of the nox promoter.
02

Design the gene construct

To make such a gene construct, the nox promoter should be cloned in front of a reporter gene in a plasmid. This could be done using standard cloning techniques. The reporter gene could be a gene that codes for a noticeable phenotype, such as fluorescence, that can be easily detected. Hence, if the nox promoter is activated due to absence of oxygen, it will stimulate the expression of the reporter gene, creating a measurable signal.
03

Transformation into yeast

The plasmid containing the construct needs to be inserted into yeast cells. This is done through transformation, which often uses heat shock to make the yeast cells temporarily permeable to DNA. Once inside the cell, the plasmid DNA gets incorporated into the yeast's own genome.
04

Functioning as an oxygen sensor

The yeast strain with the reporter could now function as an oxygen sensor. Under anaerobic conditions (low or no oxygen), the nox promoter would stimulate the expression of the reporter gene, generating a noticeable signal. If grown aerobically (in presence of oxygen), the nox promoter would not activate the reporter gene and no signal would be produced. By monitoring the output of the reporter gene, scientists could estimate oxygen levels.

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Key Concepts

These are the key concepts you need to understand to accurately answer the question.

Understanding the Nox Gene
The nox gene plays a pivotal role in yeast cell response to oxygen levels. In our scenario, the nox gene is activated under anaerobic conditions, where there is limited to no oxygen available. The ability of the nox gene to be 'switched on' in the absence of oxygen indicates it could be key for studying oxygen-related cellular processes.

By harnessing the expression pattern of the 'nox gene', scientists can create new strains of yeast that can serve as biological oxygen sensors. This is useful in a variety of research fields, including bioengineering and medicine, where understanding cellular responses to oxygen levels is essential.
The Art of Promoter Cloning
Cloning the promoter of a gene is akin to copying the command center for that gene's activation. In promoter cloning, we isolate the specific DNA sequence that controls the gene's transcription - this is the key to controlling when and where the gene is active.

The nox promoter responds to the absence of oxygen, making it an ideal candidate for cloning if we want to develop a system that indicates anaerobic conditions. The process involves using molecular techniques to identify, isolate, and then copy the promoter sequence so that it can be used in various experimental applications.
Creating Reporter Gene Constructs
A reporter gene construct serves as a tangible readout of a gene's activity. The idea is to attach a 'reportable' gene to a promoter of interest, such as the nox promoter we are studying. By doing this, whenever the nox promoter is active, the reporter gene is expressed, resulting in a measurable output.

In creating a reporter gene construct, scientists often choose reporter genes that are easy to detect, such as those encoding fluorescent proteins. Therefore, if the construct has been designed correctly, the presence or absence of light emission can directly correlate with oxygen levels in the environment of the yeast cells.
Oxygen Sensing in Yeast Cells
Yeast cells, like all organisms, respond to their environment, and oxygen is a crucial component of this. The sensing of oxygen is an internal mechanism that governs numerous cellular functions and is critical for yeast survival under varying oxygen conditions.

The nox promoter activity in relation to oxygen sensing showcases how organisms can finely tune their gene expression in response to environmental changes. By utilizing this natural mechanism, researchers can engineer yeast strains with a reporter gene that vividly indicates the oxygen levels within the cell's milieu.
Yeast Transformation Techniques
In order to study the effects of desired genetic constructs, such as our nox-promoter-reporter gene system, these constructs must be introduced into yeast cells. This is where yeast transformation comes into play. Using methods such as heat-shock or electroporation, we temporarily increase cell permeability to allow foreign DNA, like our construct, to enter the cell.

Once inside, the introduced DNA can recombine with the yeast's genomic DNA, ultimately creating a genetically modified yeast strain. As a result, we have empowered yeast cells with new capabilities, such as acting as oxygen sensors, providing a powerful tool for scientists.

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Most popular questions from this chapter

Imagine that you are interested in a protein that interacts with proteins of the cytoskeleton in human epithelial cells. Describe an experimental protocol based on the yeast two-hybrid system that would allow you to identify proteins that might interact with your protein of interest.

Yeast (Saccharomyces cerevisiae) has a genome size of \(1.21 \times 10^{7}\) bp. If a genomic library of yeast DNA was constructed in a vector capable of carrying 16 -kbp inserts, how many individual clones would have to be screened to have a \(99 \%\) probability of finding a particular fragment?

Combinatorial chemistry can be used to synthesize polymers such as oligopeptides or oligonucleotides. The number of sequence possibilities for a polymer is given by \(x^{y}\), where \(x\) is the number of different monomer types (for example, 20 different amino acids in a protein or 4 different nucleotides in a nucleic acid) and \(y\) is the number of monomers in the oligomers. a. Calculate the number of sequence possibilities for RNA oligomers 15 nucleotides long. b. Calculate the number of amino acid sequence possibilities for pentapeptides.

A vector has a polylinker containing restriction sites in the following order: Hind III, SacI, XhoI, BglII, XbaI, and ClaI. a. Give a possible nucleotide sequence for the polylinker. b. The vector is digested with Hind III and ClaI. A DNA segment contains a Hind III restriction site fragment 650 bases upstream from a ClaI site. This DNA fragment is digested with Hind III and \(C l a I,\) and the resulting Hind III-ClaI fragment is directionally cloned into the Hind III-Clal-digested vector. Give the nucleotide sequence at each end of the vector and the insert and show that the insert can be cloned into the vector in only one orientation.

Describe an experimental protocol for the preparation of two cDNA libraries, one from anaerobically grown yeast cells and the second from aerobically grown yeast cells.
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