a cloning question?

Question

You have in your freezer a complete cDNA clone for dog insulin and a piece of wolf liver. Outline a strategy for obtaining a genomic clone encompassing the promoter region of the wolf insulin gene, starting from this material and standard molecular biology reagents.
If I purify gDNA from the wolf liver, construct a genomic library and use the cDNA for dog insulin to construct a probe and then probe the genomic library, how will I be sure that the promoter will be isolated?

Answer 1

Actually, there would be no way to know whether you had actually obtained the whole promoter region from the wolf. Promoter regions are DNA sequences which allow proteins to bind to it (or prevent proteins from binding to it) which will regulate the production of that protein under different conditons. Although most promoters are upstream of the gene itself, there is no guarantee that this is true for all genes. Here is an example of a human gene with a downstream promoter:
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=150227&rendertype=abstract
If, on the other hand you are talking about the basic promoter, it would probably be more cost- and time-effective just to sequence back through the wolf promoter region starting from the known translation start site. The TATA box, the most basic promoter there is (and the only one that is truly necessary) is well defined and could be picked up by most DNA analysis software packages available today.
Once you have identified the basic promoter region, design and buy (or make) oligo primers based on your sequencing data and use PCR to amplify the region of interest and subclone it into a commercially available expression vector in front of the cDNA clone for the dog gene.
If what you are asking for are the more specialized elements of the wolf promoter, people spend years cutting and pasting bits of upstream regions to show that it increases or decreases protein production of another clone. There is no easy way to do it, unless the data has been published already (it hasn't. I checked).

Answer 2

You won't know, necessarily, until you examine the clones that you find using the probe. You can examine genomic clones for sequences upstream of the insulin gene. You'll certainly find the promotor that way.

The first answer didn't understand that you aren't talking about crossing wolves and dogs...you're asking about a molecular biology experiment...

Answer 3

Once you determine what you think to be a promoter region, you could do basic transcriptional assays- e.g. make a construct of the wolf promoter driving firefly luciferase and test it using a luminometer.

Answer 4

well this is a typical cross. one that is "in my mind" possibly unetical. you should try it out, and get the results yourself.

Answer 5

Mmmm.... wolf liver....