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.

1. Before proceeding, you wish to know whether this gene is actually expressed in humans ?Describe, in detail, 2 efficient experimental approaches to answer this question. ?Discuss whether your proposed methods measure synthesis or accumulation of the product

Answer 1

According to the information, HDM2 and HDM5 share 90% sequence similarities. Thus, it is very likely that the primers designed for one of these genes will cross-react with the other and give an amplicon product. Thus, knowing the band size of these two genes, the researcher can easily distinguish between the expression of these two genes in the target cell system.

Secondarily, in order to evaluate the nature of gene products, a knock out cell system can be created and its protein profiling can be done for investigating the nature of protein absent in the mutant as compared to the wild type. This technique can be performed by using 2-D gel electrophoresis followed by mass spectrometry to clearly identify the nature of protein.

In order to investigate if the gene is actually expressed in humans, RFLP primers of this gene can be synthesized and PCR can be performed for investigation of specific amplicon bands on the agarose gel. Presence of the amplicon bands suggests the expression of gene in the human host.

Alternatively, in situ hybridization can also be performed to investigate if the gene is expressed in the human host using chromophoric substrate-enzyme system. Positive cells suggesting expression of the gene will produce the color which can be visualized under microscope.