Genetic Analysis: An Integrated Approach (3rd Edition)
Genetic Analysis: An Integrated Approach (3rd Edition)
3rd Edition
ISBN: 9780134605173
Author: Mark F. Sanders, John L. Bowman
Publisher: PEARSON
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Chapter 16, Problem 21P

A modification of the two-hybrid system, called the one-hybrid system, is used for identifying proteins that can bind specific DNA sequences. In this method, the DNA sequence to be tested, the bait, is fused to a TATA box to drive expression of a reporter gene. The reporter gene is often chosen to complement a mutant phenotype; for example, a HIS gene may be used in a his mutant yeast strain. A cDNA library is constructed with the cDNA sequences translationally fused to the GAL4 activation domain and transformed into this yeast strain. Diagram how trans-acting proteins that bind to cis-acting regulatory sequences can be identified using a one-hybrid screen.

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When a region of DNA that contains the genetic information for a protein is isolated from a bacterial cell and inserted into a eukaryotic cell in a proper position between a promoter and a terminator, the resulting cell usually produces the correct protein. But when the experiment is done in the reverse direction (eukaryotic DNA into a bacterial cell), the correct protein is often not produced. Can you suggest an explanation?
Bacterial glucuronidase converts a colorless substance called X-Gluc into a bright blue indigo pigment. The gene for glucuronidase also works in plants if given a plant promoter region. How would you use this gene as a reporter gene to find the tissues in which a plant gene that you have just cloned is normally active? (Assume that X-Gluc is easily taken up by the plant tissues.)
Could quantitative PCR, which uses a DNA-binding dye, to show how many copies of the target DNA sequence could be used to quantify the amount of mRNA in a cell? Would you expect that a metabolically active tissue such as the liver would show more cDNA copies in such a method, compared to less metabolically active tissues such as skin cells? One reason that the types and amounts of mRNAs are quantified in different tissue types is to compare which genes are activated and which are inactive.  It used to be thought that any gene that was transcribed was automatically translated. The discovery of RNA-degrading systems shows that the real situation in cells is more complemented. Do you believe that a larger amount of mRNA of a given type, say for alpha hemoglobin in immature red blood cells is a reliable indicator that more alpha hemoglobin protein will be made in those cells?

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Genetic Analysis: An Integrated Approach (3rd Edition)

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Bacterial Genomics and Metagenomics; Author: Quadram Institute;https://www.youtube.com/watch?v=_6IdVTAFXoU;License: Standard youtube license