Publication Date

Summer 2018

Document Type

Paper

Department

Life and Environmental Sciences

First Instructor

Stefanie Otto-Hitt

Experiment Type

Reverse transcriptase (RT) PCR

Feature

TTHERM 01207660

Gene

CDC2: Homolog of the catalytic subunit of the major cell cycle cyclin-dependent kinase (cdk); studies suggest cdc2p phosphorylates HHO1; expression positively regulated by HHO1 phosphorylation during starvation, resulting in a posi

Abstract

In this experiment, the eect of constant motion on expression of the cell cycle control gene CDC2 was examined in the organism Tetrahymena thermophila. We hypothesized that CDC2 expression would decrease with a corresponding increase in movement within the Tetrahymena culture environment. The CDC2 protein is primarily used in the replication of eukaryotic DNA because of its connection to HHO1, an important factor in transcriptional regulation. If the cells were subject to vegetative growth in which there are no stressors put on the cell, then HHO1 becomes phosphorylated to prevent it from interacting with chromatin. Because CDC2 is important in facilitating the phosphorylation of the HHO1, we hypothesized that, during vegetative growth, there would likely be an increase in the expression of CDC2. Moreover, when the cell is subject to stressors there would be no need to control cell growth by phosphorylating HHO1, so CDC2 expression should decrease. To address this hypothesis, Tetrahymena thermophila were randomly assigned to a control group, which was cultured under normal lab-growth conditions, or a treatment group, which was subjected to constant movement on an orbital shaker for a period of one week. Following treatment, RNA extraction, reverse transcription, and gene-specific PCR (including gel electrophoresis) were used to analyze CDC2 expression in both the control and treatment groups. Furthermore, the growth rate of both groups was analyzed using a hemocytometer. We predicted that Tetrahymena cells subjected to constant motion would exhibit a decrease in cell growth rate and expression of CDC2.

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