MOLECULAR PROBES FOR THE ANDROGEN RECEPTOR GENE

  • Tindall, Donald J, (PI)

Project: Research project

Description

The actions of dihydrotestosterone, a hormone of central
importance in controlling the function and growth of male
reproductive tissues, such as the prostate and seminal vesicle,
appear to be mediated interaction with an intracellular
protein, the androgen receptor. Although much is known about
the nature of this protein, little is known about the gene
which codes for its mRNA or the regulation of the gene during
development and following hormonal stimulation. Our initial
efforts have been to purify and characterize the androgen
receptor from several target tissues. The focus of the current
proposal is to obtain cDNA probes which can be used to
elucidate the molecular properties of the androgen receptor
gene. In order to accomplish this goal, a monospecific
antibody will be raised against the purified androgen
receptor. The antibody will be characterized with respect to
its specificity and affinity for the androgen receptor binding
activity. A Lambdagt11 library will be screened for the
expression of androgen receptors with the use of the
anti-receptor antibodies, and a full-length cDNA clone will be
isolated and sequenced. A number of properties of the cDNA
will be examined in order to verify that it codes for the
androgen receptor. First, we will examine the tissue
distribution and content of the mRNA recognized by the cDNA by
Northern dot blot analysis and compare these levels with
receptor binding activity in order to demonstrate that there is
a correlation between the two. Both normal and testicular
feminized (Tfm) rats, and mice which have defective receptors,
will be used in these studies. Second, we will determine if
the gene coding for the cDNA is located on the X chromosome.
Third, we will utilize fusion protein analysis and amino acid
sequencing to confirm that cDNA is indeed coding for the
receptor. Fourth, we will produce mRNA from plasmids
containing a bacteriophage SP6 promoter and translate the
message in vitro to determine if it codes for the androgen
receptor. The in vitro product will be analyzed by peptide
fragmentation, steroid binding and DNA binding analyses in
order to compare it with the purified receptor. These studies
should yield new information regarding the molecular properties
of the androgen receptor. Availability of such probes will
significantly enhance our ability to study androgen receptors
at the molecular level and may help us understand
androgen-related diseases such as benign prostate hyperplasia.
StatusFinished
Effective start/end date7/1/866/30/90

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

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Molecular Probes
Androgen Receptors
Complementary DNA
Genes
Messenger RNA
Androgens
Prostate
Antibodies
Seminal Vesicles
Dihydrotestosterone
X Chromosome
Bacteriophages
Libraries
Hyperplasia
Proteins
Clone Cells
Steroids
Hormones
DNA
Growth

ASJC

  • Medicine(all)