Assays for Identifying Functional Alterations in the p53 Tumor Suppressor
Background The p53 gene is mutated in over half of all tumors, indicating its importance in the process of carcinogenesis. The gene encodes a tumor suppressing transcription factor that binds to certain DNA sequences, thereby activating a number of growth suppressing genes. There is a need to screen for mutant p53 genes that encode for certain proteins. The development of a drug that can activate p53 would be extremely useful in that it would enable physicians to acquire some control over the cell cycle.
Invention Description This invention screens for mutant p53 genes that encode for specific proteins. From screening, mutants that are more thermally stable can be isolated from the corresponding wild type. This technology can identify the portions of the normal protein that participate in the allosteric regulation of its activity. This information can be used to design a drug that can activate p53 activity, thereby giving physicians pharmaceutical control over the cell cycle. This invention utilizes a screening process to solve the problem at hand. Firstly, a library of randomly mutated p53 genes is subcloned into a plasmid and is transformed into a population of E. coli. Then, the transformed bacteria are plated on a non-inducing plate, so that each colony contains but does not yet express a single p53 mutant. Next, the colonies are transferred to a nitrocelluose filter, which is in turn overlaid onto an inducing plate. The p53 genes are induced for 2-4 hours at room temperature (or 37'C in screens for thermostable mutants). Once the genes are induced, the colonies are lysed, and the filter-bound p53 proteins are reacted with a radio-labeled, double stranded oligonucleotide probe encoding a p53 binding site. After a wash step, the bound probe is assessed by phosphorimagery. Those colonies with DNA-binding activity greater than those expressing the parental gene are isolated from the original non-inducing plate.
More effective in replacement gene therapy because thermostable p53 mutants can be isolated. No post-translational modification required to bind to the DNA ligand because self-activating p53 mutants can be isolated. Identification of proteins that are good drug targets or therapeutic agents.
Adverse conditions can be adjusted for the screening because this method is in vitro (performed outside the cell)
Direct measurement of the quantity of radio-labeled DNA ligand bound to each mutant Isolation of mutations with greater than wild-type activity Allows for differentiation between disruption of specific protein-protein interactions and global unraveling of the tertiary structure mutations of the protein The thermostable mutants would probably be more amenable to crystallographic studies than the wild-type because the three-dimensional tertiary structure is unknown.
Market Potential/Applications Applications of the screen are as follows: the co-expression of cDNA libraries (derived from normal and cancerous tissues) with the wild-type (inactive) or mutant (active) p53 gene will enable those cDNAs which encode proteins that activate the wild-type or inhibit the mutant p53 to be isolated and sequenced; and the active p53 mutant will be randomly mutated and co-expressed with certain proteins known to specifically inhibit its DNA binding activity. Active p53 mutants resistant to inhibitory proteins can then be isolated and sequenced.
IP Status One U.S. patent issued: 6,429,298
UT Researcher Andrew Ellington, Ph.D., Chemistry and Biochemistry, The University of Texas at Austin Ichiro Matsumura, Ph.D., Chemistry and Biochemistry, The University of Texas at Austin
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