1. 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 Introduction Investigated DiseasesLinked to expansion of the genetic polymorphism Expected numbers of TG repeats Cystic FibrosisYes – evidence suggests increased NO production with larger repeat size 18-36 SchizophreniaNo15-20 Parkinson’s Disease (Japanese study) No Parkinson’s Disease (Kurth’s study – preliminary data) Yes (?)28-43 A portion of the DNA sequence of the NOS1 gene (1453-1663) gccctgcgtggctactacattcagccctatccaggctcccagaaattg tcattgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtatgtgtgtgtgtgtgtgtgtg tgtgtgtttcctgatagaaaaaaaaaaatggaaattaggttatataaaag atgtatgctttggagcccagagcggctcttttaatgagggttgcgacgtctcc Figure 1. The small portion of the NOS1 promoter region containing the dinucleotide repeat (red). Numbers refer to GenBank accession sequence number U15666. Forward primer: 1455-1474hNOS1FAM 5’ - /56-FAM/CCT GCG TGG CTA CTA CAT TC-3’ Reverse primer: 1661-1642hNOS1REV 5’ – AGA CGT CGC AAC CCT CAT TA – 3’ 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. Table 1. Other research into the dinucleotide polymorphism in the NOS1 gene and whether or not the NOS1 polymorphism is linked to the diseases. Introduction of DNA into mammalian cells with calcium phosphate. DNA is introduced as a coprecipitate with calcium phosphate The DNA enters the cell by endocytosis where some of the coprecipitate escapes the endosomes/lysosomes and enters the cytoplasm. From the cytoplasm the DNA can enter the nucleus of the cell. Depending on the cell type, up to 50% of a population of cells can then express transfected genes in a transient fashion. 2 days after transfection the cells will be harvested and protein is isolated Enzyme assays using a Berthold Detection System will be performed to detect amount of luciferase activity 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. 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. 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. 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. DNA SampleNumber of samples to be tested Parkinson’s Disease patient 270 Control (No Parkinson’s Disease) 188 Table 2. The number of DNA samples, both Parkinson’s Disease patients and control DNA (no Parkinson’s Disease) provided by Dr. Judith Kurth Methodology Polymorphism Analysis PCR PCR will be performed using 2 primers which flank the region of the dinucleotide repeat and running the entire gene through the PCR. The forward primer has a fluorescent dye FAM at the 5’ end. Polymorphism size determination The samples will be sent to the Plant Microbe Genomics Facility at The Ohio State University which uses an Applied Biosystems 3700 DNA Analyzer. It can analyze DNA fragments labeled with fluorescent dyes. The analyzer can determine the quantity and size of the fragment to within 1 base. The DNA fragments are run over a column along with standards (Liz) Each individual will have 2 alleles for the gene and we are interested in seeing how far they migrate on the column – the distance that they travel on the column is directly proportion to the size of our DNA fragments Cloning The entire promoter region will be amplified by PCR from samples with a wide range of dinucleotide repeat sizes. The PCR primers used for this reaction will contain KpnI and BglI restriction enzyme sites at their 5’ ends. The PCR products will then be cloned into pGL3 luciferase reporter vector After the plasmid is introduced introded into mammalian cells – the promoter region will drive the expression of luciferase Using Luciferase to quantify gene expression Luciferase is a photoprotein produced by the North American firefly, Photinus pyralis. When expressed in mammalian cells, luciferase molecules produce luminescence in direct proportion to their number, provided that the substrate luciferin and ATP are present. In addition to luciferase constructs we will introduce betagalactosidase constructs under the control of a viral promoter as a control for transfection efficiency. Beta-galactosidase will be produced in the same amount in different transfections 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 Deletion constructs containing different lengths of the 5’-flanking sequences of the NOS1 gene were transfected into HeLa cells and NOS1 activity was analyzed. By removing this region we may in fact be removing a repressor-binding site. Figure 3. Deletion Construct data from a previous research with the NOS1 promoter. SOURCES 1.Eggers-Sedlet, B., C. Kurth, Matthias C., Lieberman, A., and Kurth, J. Nitric Oxide Synthase Gene Polymorphism Associated with Parkinson’s Disease. Unpublished – abstract cited. 31 March 1997. 2.Texereau J., Marullo S., Hubert D., Coste J., Dusser D.J., Dall'Ava-Santucci J., Dinh-Xuan, AT. Nitric oxide synthase 1 as a potential modifier gene of decline in lung function in patients with cystic fibrosis. Accessed by online database: PubMed. 15 April 2004. 3.Ying-Jay L., Shih-Jen T., Chen-Jee H., and Ding-Lieh, L. Association analysis for the CA repeat polymorphism of the neuronal nitric oxide synthase (NOS1) gene and schizophrenia. Accessed by online database: PubMed. 15 April 2004. Determination of the effect of the TG repeat on NOS transcription luciferase PXP2 1-1699 1-1799 1-1842 1-1628 1195-1842 1428-1842 1470-1842 1613-1842 1674-1842