3. The diagram below shows the DNA sequence of a gene which is expressed only in

Question

3. The diagram below shows the DNA sequence of a gene which is expressed only in the skin of the Jackalope, a diploid eukaryote. Shown is DNA sequence from close to the transcriptional start site, as well as some upstream sequence. The first nucleotide transcribed is an A, and the corresponding A on the coding strand is underlined. Two other sequences in this region are underlined.

Below the sequence is shown the structure of several pieces of DNA that I tested for their ability to initiate transcription. Construct a is the wild-type sequence and b through f either contain small deletions (b-d) or point mutations (e, f). At the right are indicated the relative levels of transcription for each construct, in Arbitrary Units.

i. For each mutant construct (b-f), indicate: what sequence element might be affected and why the expression level is changed to the extent shown. 2.5 pts

I was also able to isolate an enhancer for this gene, which could further increase the levels of expression of the promoter in Jackalope skin cells. The sequence of the enhancer is shown below.

My research has also shown that two transcription factors acting upon this gene are Mattigen and Tammigen. These proteins bind to DNA at the sequences 5’AATT3’ (for Mattigen) and 5’CCCGGG3’ (for Tammigen).

Below the sequence are the structures of five constructs: a wild-type enhancer (g), and four deletion mutants (h through k). These constructs were tested for their ability to help the promoter to increase the level of transcription. Relative levels of expression are shown, once again measured in Arbitrary Units.

ii. For each mutant construct (h-k), indicate which transcription factor’s function might be affected, and suggest why the expression level is changed to the extent shown. 2 pts

Explanation / Answer

In b, deletion has occured in GC box.

In c, deletion has occured in TATA box region.

In e, deletion of GC box and TATA box both has occured. This results in complete loss of transcription.

In e, a point mutation(transition) has occured in the TATA box region at 6th position, converting TATAAG into TATAAT, which is the most appropriate and required sequence for transcription.

In f, a point mutation (transition) has occured in TATA box region, converting TATAAG into TAGAAG, which is not the appropriate conformation of TATA box, therefore transcription has not occured (almost)

ii)

In h, i and j Mattigen function has been affected.

In k, Tammigen function has been affected.

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