Transcription of the mammalian Cytochrome P450 1A1 gene is induced by xenobiotics such as dioxin. The...

Transcription of the mammalian Cytochrome P450 1A1 gene is induced by xenobiotics such as dioxin. The mechanism of induction illustrates many steps which are typical of signal-regulated transcription factors. （16 marks）

Describe in detail the steps involved in dioxin induction of this gene
Outline an in vitro experiment you could carry out to show that Arnt is essential for the dioxin receptor to bind to the response element of the Cytochrome P450 1A1 gene.

no more detail！Thx

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Expert Solution

Answer:

Here are the following steps involved in the Cytochrome P450 1A1 gene induction by dioxin:

Firstly cell culture is prepared. For that, we can use the mouse hepatoma cell line and mouse embryonic fibroblast cell line.
Make groups of the cells with and without induction by dioxin. This chemical is an inhibitor of Cyp1a1 and Cyp1b1.
The mRNAs of Cyp1a1, Cyp1b1, and ribosomal subunit 36b4 is produced by a quantitative real-time polymerase chain reaction.
Isolate total RNA from the cell culture.
Perform reverse transcription.
Compare levels of Cyp1a1 and AHR mRNAs with a standard RNA sample obtained.
Perform Chromatin Immunoprecipitation assay for the cell cultures.
Perform Somatic cell hybrid isolation by fusing Hepa-1 cells with embryonic fibroblast cells.
Determine the DNA content of hybrid clones.
Treat all cell cultures with 5 μM 5-aza-2′-deoxycytidine with Trichostatin-A.
Perform statistical analysis.

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