Question

You are analysing RNA transcripts of one particular gene in different tissues from mice. Using a specific probe to detect the RNA transcripts in embryonic heart (EH), adult heart (AH), embryonic pituitary (EP), adult pituitary (AP), and testis (T) the following pattern is observed:

ЕН АН ЕР АР Т 1.4 kb 1.2 kb

What would you conclude about the gene based on the results with AH RNA? Explain the possible underlying molecular mechanism.

How would you explain the results with testis RNA?

Explain the possible underlying molecular mechanism. Which enzyme complex is important? What does this enzyme complex recognize and what molecule class is mediating the catalytic process?

Answer 1

Sequence-specific small interfering RNA (siRNA) duplexes can be used for gene silencing in mammalian cells and as mechanistic probes for determining gene function.The transfection of siRNAs for the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) mRNAs in MCF-7 breast cancer cells resulted in a 60 to 80% decrease in levels of AhR and ARNT proteins in whole-cell extracts and decreased binding of nuclear extracts to 32 P-labeled dioxin-responsive element. siRNA for the AhR also decreased 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced CYP1A1 protein, CYP1A1-dependent activity, and luciferase activity in cells transfected with an Ah-responsive construct. 17 beta-estradiol (E2) induces proliferation of MCF-7 cells through enhanced G0/G1 --> S phase progression, and this response is inhibited in cells cotreated with E2 plus TCDD. The effects of TCDD on E2-induced cell-cycle progress were partially blocked in MCF-7 cells transfected with siRNA for AhR. The results also indicated that siRNA-dependent decreases in AhR protein in MCF-7 cells were accompanied by increased G0/G1 --> S phase progression, suggesting a growth-inhibitory role for the endogenous AhR.