P1247176259FOgZE

1
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. Josephs Hospital
Medical Center, Phoenix, Arizona
Using Luciferase to quantify gene expression

• 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

Introduction
Parkinsons 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. Parkinsons 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
Parkinsons Disease patients and control DNA (no
Parkinsons Disease) provided by Dr. Judith Kurth

Methodology

• 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

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.

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.

• 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

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.

Determination of the effect of the TG repeat on
NOS transcription

• 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

1674-1842
1613-1842
1470-1842
1428-1842
1195-1842
1-1628
1-1699
1-1799
1-1842
PXP2
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.

• SOURCES
• Eggers-Sedlet, B., C. Kurth, Matthias C.,
  Lieberman, A., and Kurth, J. Nitric Oxide
  Synthase Gene Polymorphism Associated with
  Parkinsons Disease. Unpublished abstract
  cited. 31 March 1997.
• 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.
• 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.

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. Kurths
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.