• Wieben, Eric D (PI)
  • David, Chella S (PI)
  • Spelsberg, Thomas C (PI)
  • Tindall, Donald J (PI)
  • Spelsberg, Thomas C (PI)
  • Pendergast, F. (PI)
  • Tindall, Donald J. (PI)
  • Roche, Patrick (PI)
  • Pendergast, F. (PI)
  • Penniston, John (PI)
  • Ryan, Robert (PI)
  • Roche, Patrick (PI)
  • Toft, David (PI)
  • Tindall, Donald J. (PI)
  • Tindall, Donald J. (PI)
  • Wieben, Eric D (PI)
  • Tindall, Donald J (PI)
  • David, Chella S (PI)
  • Wieben, Eric D (PI)
  • Tindall, Donald J (PI)

Project: Research project

Project Details


It is long range goal of this project to determine the composition,
function and location of the nuclear acceptor sites (nuclear binding sites)
of the avian progesterone receptor (PR). Two, and possibly other,
candidate chromatin acceptor proteins for PR, termed Receptor Binding
Factors (RBF), have been identified. As these proteins are removed from
the chromatin (along with other proteins), a loss of the specific PR
binding sites for PR is observed. When bound to avian DNA, both impure and
pure fractions of these proteins generate high affinity, saturable,
receptor-dependent binding of the PR to DNA. One of these proteins, RBF-1,
has been purified over 105x fold to apparent homogeneity. It has been
partially sequenced and monoclonal antibodies (MAb) prepared against it.
RBF-1 is a unique 10 kDa, hydrophobic protein with a pI ~5.5 and is species
and tissue specific. The RBF-1 and the PR nuclear binding sites were both
localized to the oviduct nuclear matrix using MAb in Western blot analyses
and cell-free binding assays respectively. The RBF-1 activity has been
distinguished from a second activity, RBF-2, which is more tightly bound
to the DNA than the RBF-1 and has been under investigation for some time as
a component of the nucleoacidic protein (NAP). RBF-2 does generate
specific PR binding sites and is also suspected to be associated with the
nuclear matrix. However, no proteins in the crude RBF-2 preparations are
recognized by the anti-RBF-1 antibodies. Recent studies using Southwestern
blot (and DNA gel shift) analyses indicate that RBF-1 has a high affinity
for specific sequences in the matrix DNA. Preliminary evidence also
suggests that these sequences may reside in or near the steroid regulated
genes but not the nonregulated genes. This project proposes to further
characterize RBF-1 by completing its amino acid sequence, confirm its
intracellular/nuclear location using in situ techniques, determine its
receptor specificity and its half-life in primary oviduct cell cultures,
and assess its role in generating the high affinity class of PR binding
sites. Identification of the DNA element to which the RBF-1 binds will be
attempted using Southwestern and DNA gel shift assays. Its sequence will
be compared to those of known steroid response elements, as well as to
sequences in or around steroid regulated genes, with emphasis on the rapid
steroid regulated c-myc and c-jun proto-oncogenes. The specific bases
within the element to which the RBF-1 binds, and the additional bases to
which the receptor (PR) itself may bind, will be examined. Lastly, the
role of this element and/or its flanking domains, as steroid response
element, will be examined. The isolation of the cDNA and genomic sequences
of the RBF-1 protein and the analyses of the biological function of the
protein will be pursued. Attempts to purify and characterize the RBF-2
will made if time permits.
Effective start/end date5/1/753/31/02


  • Medicine(all)