luciferase. Investigation of a dinucleotide repeat in the human NOS1 gene Jon Altizer 1 , Yu Cao 1 , Janice Kurth 2 , Terrie Rife 1 1. James Madison University, Harrisonburg, VA 2. Barrow Neurological Institute, St. Joseph’s Hospital & Medical Center, Phoenix, Arizona.
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Investigation of a dinucleotide repeat in the human NOS1 geneJon Altizer1, Yu Cao1, Janice Kurth2, Terrie Rife11. James Madison University, Harrisonburg, VA 2. Barrow Neurological Institute, St. Joseph’s Hospital & Medical Center, Phoenix, Arizona
Using Luciferase to quantify gene expression
Parkinson’s disease (PD) is a chronic progressive neurological disease that affects a small area of nerve cells (neurons) in an area of the brain known as the substantia nigra. PD most likely results from a combination of environmental and genetic factors. Our lab is concerned with a genetic polymorphism in the promoter region of the NOS1 gene. An expansion of a dinucleotide repeat (TG repeats) in this region of the gene has been suggested to be linked to PD. Parkinson’s patients have fluctuations in the level of NOS1 protein during the duration of the disease. In the beginning stages of PD the levels of NOS1 increase, but when looking at end-stage PD patients the NOS1 level has decreased. NOS I is an enzyme that catalyzes the conversion of arginine to citruline, which is a process that produces the free radical nitric oxide (NO). Transcriptional regulation of NOS1 is critical because large amounts of NO can cause neurodegeneration.
Table 2. The number of DNA samples, both Parkinson’s Disease patients and control DNA (no Parkinson’s Disease) provided by Dr. Judith Kurth
Figure 1. The small portion of the NOS1 promoter region containing the dinucleotide repeat (red). Numbers refer to GenBank accession sequence number U15666.
Table 3. The DNA sequence of the forward and reverse primers utilized in the PCR procedure. The fluorescent dye FAM is utilized when determining the size of the dinucleotide repeat.
Several research groups have looked at this TG repeat and its correlation to other diseases. One group in China looked at the TG repeat and whether or not a patient was at higher risk of developing Schizophrenia. This group concluded that there was no correlation between having this TG repeat and a patients’ susceptibility to develop Schizophrenia. Another research group looked at the correlation between having a dinucleotide repeat and patients with Cystic Fibrosis (CF). This research group has introductory data showing a positive correlation between having the dinucleotide repeat and susceptibility to developing Cystic Fibrosis.
Figure 3. The chemical formula showing how light is produced (luminescence) when luciferin and ATP are present – the amount of light produced is directly proportional to the number of luciferase molecules.
Previous research with this NOS1 promoter
Determination of the effect of the TG repeat on NOS transcription
Figure 3. Deletion Construct data from a previous research with the NOS1 promoter.
Table 1. Other research into the dinucleotide polymorphism in the NOS1 gene and whether or not the NOS1 polymorphism is linked to the diseases.
The DNA being utilized in our research has been provided by Dr. Judith Kurth. She is now a CEO of a company and is not able to continue her study. She has provided us with her control samples as well as her PD patient samples. Based upon her preliminary data she has shown a positive correlation between having an elongated dinucleotide repeat and the predisposition for PD.Our goal includes finishing Dr. Kurth’s research and determining if having an elongated dinucleotide repeat effects NOS1 transcription.
Figure 2. The pGL3-Basic vector which will be utilized for our cloning procedure. The vector will be opened at the KpnI and BglII restriction enzyme sites.