lex A is an E. coli gene whose product regulates the transcription of certain ta
ID: 30703 • Letter: L
Question
lex A is an E. coli gene whose product regulates the transcription of certain target
genes that are located at different positions in the bacterial chromosome.
Identified target genes appear to be involved in DNA repair and recombination.
Under normal conditions, the LexA protein binds near and represses
transcription of target genes; however, when the cell experiences significant
DNA damage caused by environmental mutagens, the LexA protein is
inactivated and transcription of the target genes occurs. Your research objective
is to identify all of the target genes repressed by the LexA protein. (Assume that
you have access to a DNA microarray consisting of sequences representing each
of the genes in the E. coli genome.) As the starting point for your work, you
isolate a lexA mutant strain that produces no functional LexA protein.
a) Briefly describe an experiment that uses a functional genomics approach to identify all
of the LexA target genes.
b) Give the basic steps of the experiment and show how it
would allow you to achieve your objective
Explanation / Answer
a)
To know the Lex A protein target sequences first Lex A mutant strain must be isolated. For this, Replica plating technique can be employed. The master plate containing Lex A+ and Lex A- is selected and the test plate should contain the medium that supports the growth of only Lex A- strains. A velvet cloth is pressed on to master plate is again pressed on the test plate and in cubated. After incubation the cell that contain nonfunctional Lex A (Lex A-) can be isolated.
Once the nonfunctional Lex A mutant strain is isolated then DNA microarray analysis can be used to detect the target sequences of Lex A protein. For this, immunoprecipitation of Lex A protein can be done. Then, the protein targeting DNA sequences can be identified throughout the genome through the hybridization of DNA fragments.
b)
The experimental design is as follows:
The DNA is extracted from Lex A- strains and placed on to polyagarose or silica surface.
A target probe is applied, so that hybridization take place.
First the target protein immunoprecipitated with flourescent antibody.
Then the protein targeting sequences can be detected through hybridization with oligonucleotide probes.
The DNA chip can be scanned to find the number of target sequences of Lex A protein throughout the genome.
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