Identification of Interacting Proteins Via a Yeast Genetic Screen

Location

CSU 253/254/255

Start Date

12-4-2004 1:45 PM

End Date

12-4-2004 3:15 PM

Student's Major

Biological Sciences

Student's College

Science, Engineering and Technology

Mentor's Name

Marilyn Hart

Mentor's Department

Biological Sciences

Mentor's College

Science, Engineering and Technology

Description

Actin, a component of all eukaryotic cells, contributes to cell motility, shape and organization. In striated muscle, actin capping protein (CP) binds to the barbed end of the actin filament at the Zline, directing and maintaining proper organization. CP is a heterodimer composed of alpha and beta subunits. Three isoforms of the beta subunit have been identified: betal (pi), beta! (p2), betaS (p3). The beta isoforms are produced via alternative splicing of one gene and share 90% sequence identity. Previous studies revealed that the beta isoforms have distinct localizations in cardiac myocytes with pi localizing to the Z-line and p2 localizing to the cell periphery and intercalated discs. In transgenic studies, the pi isoform was unable to functionally replace the p2 isoform and vice-versa. These studies suggest that the unique function of the beta isoforms may be due to interactions with additional cellular components. The purpose of this research is to identify proteins that interact with the pi and p2 isoforms of actin CP using a yeast two-hybrid genetic screen. The genetic screen relies upon identifiable gene expression induced by protein interactions. The necessary plasmid constructs have been generated and their orientations confirmed. Western blot analysis revealed that the constructs express the pi and p2 proteins. A small scale preliminary screen is underway.

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Apr 12th, 1:45 PM Apr 12th, 3:15 PM

Identification of Interacting Proteins Via a Yeast Genetic Screen

CSU 253/254/255

Actin, a component of all eukaryotic cells, contributes to cell motility, shape and organization. In striated muscle, actin capping protein (CP) binds to the barbed end of the actin filament at the Zline, directing and maintaining proper organization. CP is a heterodimer composed of alpha and beta subunits. Three isoforms of the beta subunit have been identified: betal (pi), beta! (p2), betaS (p3). The beta isoforms are produced via alternative splicing of one gene and share 90% sequence identity. Previous studies revealed that the beta isoforms have distinct localizations in cardiac myocytes with pi localizing to the Z-line and p2 localizing to the cell periphery and intercalated discs. In transgenic studies, the pi isoform was unable to functionally replace the p2 isoform and vice-versa. These studies suggest that the unique function of the beta isoforms may be due to interactions with additional cellular components. The purpose of this research is to identify proteins that interact with the pi and p2 isoforms of actin CP using a yeast two-hybrid genetic screen. The genetic screen relies upon identifiable gene expression induced by protein interactions. The necessary plasmid constructs have been generated and their orientations confirmed. Western blot analysis revealed that the constructs express the pi and p2 proteins. A small scale preliminary screen is underway.