Yeast 2 Hybrid Protein-Protein Interactions in DSCAM
W37
Abstract
Yeast 2 Hybrid Protein-Protein Interactions in DSCAM
Nya Fouche, Austin Kindall, Melissa Clemons, Dr. Peter Fuerst
Neural development requires the coordination of a large number of cell surface receptors. While the function of these receptors is beginning to be understood, how they transduce signals into the cell is largely unexplored. In this study, protein-protein interactions between the down syndrome cell adhesion molecule (DSCAM) and candidate signaling molecules were studied by using a Lex-A Yeast 2 Hybrid system. EGY48 of the Saccharomyces cerevisiae yeast species were used to screen a library of mouse brain cDNAs using the c-terminus of Dscam. The yeast were transformed to contain the prey plasmid with a protein library that contains unknown set of potential interactors, and grown on culture containing dextrose and amino acid media. The colonies were then stamped to selective media containing galactose, which is responsible for inducing the library plasmid and would allow growth on media lacking leucine if protein-protein interactions were occurring. The large colonies that have grown from the selective media were thought to have protein-protein interactions between the bait plasmid and the prey plasmid. The large colonies were then isolated to form single colony plates for further study. 4 single colonies were streaked to dextrose-based culture grid plates lacking the amino acids histidine and tryptophan. The grid colonies were streaked on selective media without leucine, our reporter protein, in order to analyze the role of galactose in gene expression. The colonies that did not grow on these plates were further studied with serial dilutions and PCR in order to determine the biology of the yeast DNA. Sequencing of the inserts identified several candidate interactors that may be responsible for DSCAM signaling.
This project was funded in part by the National Science Foundation REU Site award No. 1757826
Yeast 2 Hybrid Protein-Protein Interactions in DSCAM
Yeast 2 Hybrid Protein-Protein Interactions in DSCAM
Nya Fouche, Austin Kindall, Melissa Clemons, Dr. Peter Fuerst
Neural development requires the coordination of a large number of cell surface receptors. While the function of these receptors is beginning to be understood, how they transduce signals into the cell is largely unexplored. In this study, protein-protein interactions between the down syndrome cell adhesion molecule (DSCAM) and candidate signaling molecules were studied by using a Lex-A Yeast 2 Hybrid system. EGY48 of the Saccharomyces cerevisiae yeast species were used to screen a library of mouse brain cDNAs using the c-terminus of Dscam. The yeast were transformed to contain the prey plasmid with a protein library that contains unknown set of potential interactors, and grown on culture containing dextrose and amino acid media. The colonies were then stamped to selective media containing galactose, which is responsible for inducing the library plasmid and would allow growth on media lacking leucine if protein-protein interactions were occurring. The large colonies that have grown from the selective media were thought to have protein-protein interactions between the bait plasmid and the prey plasmid. The large colonies were then isolated to form single colony plates for further study. 4 single colonies were streaked to dextrose-based culture grid plates lacking the amino acids histidine and tryptophan. The grid colonies were streaked on selective media without leucine, our reporter protein, in order to analyze the role of galactose in gene expression. The colonies that did not grow on these plates were further studied with serial dilutions and PCR in order to determine the biology of the yeast DNA. Sequencing of the inserts identified several candidate interactors that may be responsible for DSCAM signaling.
This project was funded in part by the National Science Foundation REU Site award No. 1757826