You are a researcher at a small biotech company and your company has just obtain
ID: 176099 • Letter: Y
Question
You are a researcher at a small biotech company and your company has just obtained the license for use of a human GENOMIC DNA fragment putatively encoding a potentially novel protein, which is thought to regulate p53, the known tumor supressor protein. The scientists who originally cloned this GENE fragment HDM5 "claim" that HDM5 shares 90% DNA sequence homology with one of the HDM2 genes (refer to the review Levine & Oren, 2009). They propose that HDM5 may have HDM2-like properties and may be involved in regulating cell proliferation, and thus a good target to potentially develop as a cancer therapy. Your company has asked you to characterize the gene and gene products, as well as to provide an opinion as to its potential human therapeutic uses.
Answer the following questions regarding your proposed characterization of this potentially novel gene and gene product.
1. (5 points) Your first question: how does a genomic DNA fragment differ from a cDNA fragment?
2. Might you expect to find evidence of retroposons or transposons in your gene? If so, which type would be most prevalent? Explain your answers.
3. If this gene fragment HDM5 shares 90% DNA sequence similarity with HDM2 human genes, would it necessarily share 90% amino acid sequence similarity? Why or why not? Explain your reasoning thoroughly, and include a definition of the degeneracy of the DNA code.
4. Your gene would need to be transcriptionally active in order for mRNA to be transcribed. A transcriptionally active gene compared with a transcriptionally INactive gene would be expected to (you may choose more than one of the following):
a. contain acteylated histones
b. contain unacetylated histones.
c. be sensitive to DNase I.
d. be resistant to DNase I.
Explain why selected, or did not select, each option.
6. You know that multiple mRNAs can arise from a primary transcript by a number of mechanisms. You will be looking for these possibilities. Multiple mRNAs can arise from a primary transcript by use of which of the following (you may choose more than one):
a) alternative splicing
b) alternative promoters
c) alternative ribosome binding sites
d) alternative poly A sites
e) exon skipping, mutually exclusive exon use, intron retention
Explain why you either selected, or did not select, each option.
7.
After convincing yourself that this gene is expressed in humans, you now wish to determine the potential function of this protein by preparing cells in culture which express the protein.
a. How would you construct the expression plasmid (what elements would you include?).
b. Would you use the genomic fragment or a cDNA, and how would you obtain this material for insertion to your plasmid? Explain.
c. How would you transfect this plasmid into cells? Include an explanation of the method you would use to introduce the DNA and
d. What cell types would you use, and why?
e. Would your proposed preparation of transfected cells be a primary cell population, a cell strain or a cell line? Explain.
f. How would you confirm expression of your cDNA? Explain.
8. Before proceeding, you wish to know whether this gene is actually expressed in humans (your target market for therapeutic development).
Describe, in detail, 2 efficient experimental approaches to answer this question.
Discuss whether your proposed methods measure synthesis or accumulation of the product.
Explanation / Answer
1.
cDNA fragments has the only coding sequence, so expression is easy.
2. Transposable elements are the long DNA sequences in eukaryotes and prokaryotes that behave as mobile genetic elements. Thes elements form a large part of the genome of many species and they transpose by the mechanism that invlove the synthesis of DNA followed by random integration at random site. Retrotransposons are present in the eukaryotic genome which can synthesize reverse transcriptase enzyme and can transpose DNA from RNA intermediate.
The p53 gene acts through conserved mechanism to control transposon movement. This gene contains both the retroposons and the transposable elements but more transposons will be found.
Genomic DNA fragments cDNA fragments DNA is extracted from the cells and are digested by restriction endonucleases to form genomic DNA fragments. The cDNA fragemnts are created from RNA template by using reverse transcriptase enzyme. Genomic DNA fragments are inserted into vectors to form genomic library. cDNA fragments are collected to form cDNA library. Genomic DNA library contains the complete genome i.e exons and introns. cDNA library contains no introns and no DNA sequence. Genomic DNA fragments have both coding and non-coding sequences, so the expression is difficult.cDNA fragments has the only coding sequence, so expression is easy.
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