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.
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
