Size 3. You read a report that depressed mice have less of the \"Hpy mRNA: Norma

Question

Size 3. You read a report that depressed mice have less of the "Hpy mRNA: Normal Depressed bases protein activity in their brains than normal mice have in their brains Source 10,000 You hypothesize that Hpy gene expression is regulated at the level of transcription. You test this hypothesis by isolating mRNA from 8,000 neurons obtained from normal and depressed mice, separating them on 6,000 a gel and performing a Northern blot using a probe to the large first 4,000 exon of the Hpy gene. Is the result pictured at the left consistent with 2,000 your hypothesis? Why or why not? 3a. To further explore the transcriptional regulation of the Hpy gene, you need its promoter. How can you obtain this DNA? 3b. Having cloned the Hpy promoter, you fuse it to a beta-galactosidase reporter gene and transfect the fusion into a neuronal cell line and measure the beta-gal activity. You use the reporter to map cis-acting elements in the Hpy promoter by deletion mutagenesis of the indicated regions upstream of the Hpy transcription start site. The results are shown in the table. Schematic Promoter DNA Expression

Explanation / Answer

3.

Yes. It is consistent with the hypothesis.

Intensity of the band corresponding to Hpy gene is less in depressed mice compared to that in normal mice.

3a.

We need to first to find the transcription start site of Hpy gene. It can be obtained from UCSC browser. In UCSC browser, we have to search Hpy gene and find principle transcript and its sequence. First base in mRNA sequence is transcription start site. Promoter will be usually 35 bp upstream of transcription start site.

If we want to clone promoter region of the gene, usually we include a region up to 300 bp upstream of promoter and up to transcription start site. We design primers to amplify this region and do PCR amplification using muse genomic DNA as template.

3b.

Region A must be promoter because it is 35 upstream of transcription start site and its deletion lead to complete lack of reporter activity.

Region B has no role in transcription regulation. Its deletion has no effect on transcription.

Region C and D are perhaps promoter-proximal elements (PPE). PPE are are relatively short (15–30 base pairs in size), are located within the first 200 base pairs upstream of the transcription start site. Deletion of these region inhibited transcription.

Note: C and D would not be enhancers. Enhancers are usually are 100–200 base pairs long. Therefore, they are much longer to be accommodated in to the region between -75 and -120.

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