REGULATION OF BETA-AMYLOID GENE PROMOTER IN CELL TYPES

Project: Research project

Description

Beta-amyloid is the major proteinaceous component of the amyloid deposits
that accumulate extracellularly in the brain of patients with Alzheimer's
disease (AD). Beta-protein is derived from a larger amyloid protein
precursor (APP) which is encoded by a gene on human chromosome 21. The
APP gene encodes multiple transcripts for a family of secreted proteins
that are generated by alternate splicing. The APP gene is differentially
expressed in all major tissues. The hypothesis is that the upregulation
of the promoter activity of the APP gene resulting in its enhanced
transcription can set in motion the series of events culminating in
amyloid deposits. The long term goal is to study the transcriptional
control of APP gene. The specific aims are: 1) to examine the
transcription at the APP promoter in different cell types and study the
effect of different factors on the promoter; 2) to characterize the
regulatory regions (cis-acting) of the APP promoter and identify the cell
type-specific (trans-acting) factor(s) that might modulate the
transcription of APP; 3) to study the effect of homeobox gene products on
the promoter and 4) to determine whether there is a mutation in the APP
gene promoter that is isolated from persons with familial Alzheimer's
disease (FAD). Expression studies of the APP promoter will be done by constructing
chloramphenicol acetyltransferase (CAT) based promoter plasmids with a
0.6-3.2kb fragment of the APP promoter. The strength of the promoter is
measured by assaying CAT activity and determining the size of transcripts
by ribonuclease protection assay in different cell extracts. Cell lines
are of neuronal, glial, and chromaffin origin. The effect of
interleukins, phorbol esters and heat shock treatment on the promoter
will be studied in different stably transfected cells. To characterize
the regulatory domains on the APP promoter, a progressive deletion from
the 5' -end is made by limited nuclease digestion and using polymerase
chain reaction (PCR). These deleted fragments are ligated to the
reporter gene and the promoter activity is measured in different
cell-types. Such experiments could detect the presence of an
enhancer/silencer sequence in the promoter. The site-directed
mutagenesis will be employed to determine the specificity of DNA
regulatory elements of the APP promoter. The identification of any
cell-specific factor(s) that might affect the promoter activity will be
performed by gel shift assay using the appropriate promoter fragment and
proteins from cell extracts. The promoter region of the APP gene
contains 5 potential binding sites for a homeobox gene (Hox-1.3).
Homeobox gene products act as transcriptional factors. We will examine
the role of homeobox gene products with a cotransfection model using
plasmid constructs with activator gene (Hox-3.l) and target gene (APP).
To determine a mutation within the promoter region isolated from persons
with presenile cases of FAD, two flanking oligonucleotide primers that
span the O.8kb promoter will be synthesized. The flanked promoter from
different sources will be amplified by PCR technique and then sequenced
to locate a mutation.
StatusFinished
Effective start/end date9/1/922/28/99

Funding

  • National Institutes of Health
  • National Institutes of Health: $153,791.00
  • National Institutes of Health
  • National Institutes of Health: $164,620.00

Fingerprint

Amyloid
Genes
Homeobox Genes
Amyloid beta-Protein Precursor
Cell Extracts
Genetic Promoter Regions
Mutation
Acetyltransferases
Chromosomes, Human, Pair 21
DNA Primers
Trans-Activators
Alternative Splicing
Human Chromosomes
Phorbol Esters
Ribonucleases
Plasmids
Neuroglia
Digestion
Shock
Hot Temperature

ASJC

  • Medicine(all)