You are interested in the U. (50 points) miRNA1. The gene by overexpressing TF4

Question

You are interested in the U. (50 points) miRNA1. The gene by overexpressing TF4 and miRNA1 in these cells. Finaly, you also introduce Because the siRNA tube each experimental condition you extract RNA or Protein extract from the Western Blot. The Northerm Blot probe and the Western Blot antibody Gene A model (Exon lengths given in nt) model for Gene A is shown below. You perform several e you assume is regulated by transcription factor 4 (TF4) and You perform several experiments in cultured ABC cells was given to you by a collaborator, you don't know its sequence or where it binds For cells and perform a Northern and a antibody detect all Gene A isoforms 500 700 300 Answer the questions below and explain your answers. All questions blots. (30 words each question) require you to explain your answer by describing the bands you see on the Northern Blot Gene A by itself? 1600T 3 1500 1400- A.) Does the ABC cell line express B.)Does TF4 activate Gene A transcription? 1200 1100- 400- Western Blot C.) How many different transcript isoforms of Gene A do you detect? What i their likely exon composition? ABC cells+++ TF4 miRNA1 siRNA! + D.) What is the effect of miRNA1 on Gene A RNA and Protein levels? Does it affect all isoforms equally? E.) What is the effect of siRNA1 on Gene A RNA and Protein levels? Does it affect all isoforms equally F) Based on its effect, where is siRNA1 likely to bind Gene A isoforms?

Explanation / Answer

A) ABC cell line does not express gene A by itself. This is because as per the northern blot and western blot pictures in the question, gene A mRNA and protein are not expressed in the northern blot and western blot respectively in ABC cell line alone.

B)Yes, TF4 activates gene A transcription. This is because gene A mRNA and protein are expressed and the band is seen in the northern blot and western blots respectively.

C) Two different transcripts are observed one with protein length 200 amino acids and other with length 300 amino acids.

D)miRNA will inhibit translation. Hence no protein band is seen in the western blot in the presence of miRNA. However, miRNA does not inhibit transcription as mRNA band is seen in the northern blot.

It effects all isoforms equally

E) siRNA will inhibit transcription and translation of one isoform hence 300bp exon is not expressed and no 1500 bp mRNA band is observed in the southern blot.

F)siRNA binds to the 300bp exon in the gene A isoforms hence its mRNA is not expressed.

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