Detecting Protein-Protein Interactions with RNA Helicase A (RHA-1) in Caenorhabditis elegans A Yeast Two-Hybrid Screen Assay

Brent Gardner

Date of Award

2007

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Keywords

C. elegans, RNA Helicase A, Two-Hybrid

Area of Concentration

Biochemistry

Abstract

RNA Helicase A (RHA-1) is a highly conserved protein in many organisms, with orthologs in nematodes, fruit flies, mice, cows, and humans. In C. elegans, a homozygous deletion of the rha-1 gene results in stunted gonadal development, and RHA-1 is known to be involved in a variety of cellular processes from transcription regulation to nuclear transport. RHA-1 is also known to interact with BRCA-1 and Creb Binding Protein; yet, despite knockout and protein-protein interaction studies, not many other biochemical mechanisms are known concerning RHA-1 in C. elegans. The purpose of this study was to find other protein-protein interactions with RHA-1 in C. elegans using yeast two-hybrid screening methods. Once protein-protein interactions are found, more in-depth mechanisms and biological processes surrounding RHA-1 in C. elegans, and in other organisms including humans, may be inferred. The yeast two-hybrid method derives its selective screening power from the genetic separation of a transcription activator (GAL4) into its two main domains: the DNA-binding domain (DBD) and the transcription activation domain (TAD). The DBD is genetically attached to RHA-1, the bait, and the TAD is then genetically tagged to random C. elegans library proteins, the preys. When a resulting protein-protein interaction occurs, the DBD and TAD are again brought back together to constitute a complete GAL4 transcription activator once more. Resulting transcription activation induces expression of HIS3 and lacZ reporter genes, which may be readily detected. From the twelve prey proteins pulled from the assay screen, none were found to be true positive results. Such amounts of false positives is an inherent problem with two-hybrid studies. Future two-hybrid studies are recommended in which the bait would contain only specific domains of RHA-1.

Rights

This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.

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