Effect of EGTA on SIT1 Scramblase Gene Expression and Cell Growth in Tetrahymena thermophila

Abstract

Scramblase is an enzyme that facilitates the movement of newly synthesized phospholipids from the cytosolic side to the extracellular side of the lipid bilayer. This process is vital for cell membrane repair and growth. In Tetrahymena thermophila, the gene SIT1 encodes for the Scramblase protein, whose functionality is Ca2+-dependent. In this experiment, the concentration of accessible Ca2+ ions was decreased in order to observe whether the change had an aect on the expression of SIT1 and cell growth. It was hypothesized that expression of the SIT1 gene would increase, while cell growth would decrease. To carry out the experiment, Tetrahymena thermophila were randomly separated into either a control or experimental group. The control groups were exposed to conditions with no Ca2+ inhibitor, while the experimental groups were exposed to EthyleneDiamineTetraacetic Acid (EDTA). EDTA is a commonly used chelating agent that inhibits the function of calcium ions. After exposing the experimental group to a single dose of EDTA over the course of one week, RNA extraction, reverse transcription, and semi-quantitative gene-specific PCR were performed on both the experimental and control groups to measure expression of SIT1. Cell growth was also measured throughout the week of culturing by counting cells on a hemocytometer. Because the addition of EDTA results in a deficiency of accessible Ca2+ ions, it was predicted that SIT1 gene expression would increase due to decreased productivity of available Scramblase proteins, and that cell growth would decrease due to the inability of these Tetrahymena to repair their membranes.