Characterization of Proteins that Interact with the Alpha Subunit of Actin Capping Protein

Location

CSU Ballroom

Start Date

21-4-2008 1:00 PM

End Date

21-4-2008 3:00 PM

Student's Major

Biological Sciences

Student's College

Science, Engineering and Technology

Mentor's Name

Marilyn C. Hart

Mentor's Department

Biological Sciences

Mentor's College

Science, Engineering and Technology

Description

Actin plays an important role in many cellular functions including muscle contraction, cytokinesis, cell motility, maintenance of size and shape and cell signaling. Actin is regulated by various accessory proteins including actin capping protein (CP). The heterodimeric CP, which is composed of both an alpha (α) and beta (β) subunit, regulates actin by binding tightly to the barbed end of the actin filament. Higher organisms contain three α and three β subunits. I hypothesized that the α isoforms have unique cellular and biochemical roles and therefore interact with different cellular proteins. A yeast two hybrid screen was employed using a murine embryonic cDNA as the prey and either α1 or α2 as the bait. The objective of the screen was to identify protein interactions between α1 or α2 and other structural or regulatory proteins. Five interacting clones were identified from the α1 screen and seven interacting clones were identified from the α2 screen. Sequence analysis confirmed the identity of four of the α2 clones. To characterize the remaining clones, I isolated the plasmid DNA from the yeast cells by growing the yeast on selective drop out media and utilizing the Clontech Yeast Plasmid Isolation kit, and have confirmed the integrity of the DNA. The plasmid DNA was transformed into KC8 chemically competent cells. The QIAGEN miniprep protocol was utilized to isolate the plasmid DNA from KC8 cells. Polymerase Chain Reaction (PCR) was performed to amplify the plasmid insert, characterizing insert size. DNA sequence analysis confirmed the identity of interacting clones.

This document is currently not available here.

Share

COinS
 
Apr 21st, 1:00 PM Apr 21st, 3:00 PM

Characterization of Proteins that Interact with the Alpha Subunit of Actin Capping Protein

CSU Ballroom

Actin plays an important role in many cellular functions including muscle contraction, cytokinesis, cell motility, maintenance of size and shape and cell signaling. Actin is regulated by various accessory proteins including actin capping protein (CP). The heterodimeric CP, which is composed of both an alpha (α) and beta (β) subunit, regulates actin by binding tightly to the barbed end of the actin filament. Higher organisms contain three α and three β subunits. I hypothesized that the α isoforms have unique cellular and biochemical roles and therefore interact with different cellular proteins. A yeast two hybrid screen was employed using a murine embryonic cDNA as the prey and either α1 or α2 as the bait. The objective of the screen was to identify protein interactions between α1 or α2 and other structural or regulatory proteins. Five interacting clones were identified from the α1 screen and seven interacting clones were identified from the α2 screen. Sequence analysis confirmed the identity of four of the α2 clones. To characterize the remaining clones, I isolated the plasmid DNA from the yeast cells by growing the yeast on selective drop out media and utilizing the Clontech Yeast Plasmid Isolation kit, and have confirmed the integrity of the DNA. The plasmid DNA was transformed into KC8 chemically competent cells. The QIAGEN miniprep protocol was utilized to isolate the plasmid DNA from KC8 cells. Polymerase Chain Reaction (PCR) was performed to amplify the plasmid insert, characterizing insert size. DNA sequence analysis confirmed the identity of interacting clones.

Recommended Citation

Raver, Ryan. "Characterization of Proteins that Interact with the Alpha Subunit of Actin Capping Protein." Undergraduate Research Symposium, Mankato, MN, April 21, 2008.
https://cornerstone.lib.mnsu.edu/urs/2008/poster-session-B/16