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As a geneticist, I have a persistent interest
in the evolution of gene families and how individual family members assume
different and varied functions within developmental programs. A major focus
of our laboratory has been an examination of the organization and evolution
of gene families, and the programmed activation of evolutionarily related
genes during invertebrate development. Over the past five years,
our laboratory has been characterizing the structure and expression of
genes involved in invertebrate steroid hormone signaling, a response
mediated by the ecdysteroid receptor, a member of the nuclear receptor
(NR) gene family.
In animals, the steroid hormones have long been recognized as playing important
roles in regulating many physiological processes, from calcium balance
to growth and development. Similarly, vitamin A and its hormone-like
derivatives, the retinoids, are essential for normal development, and either
retinoid deficiency or excess can be profoundly damaging, causing a variety
of pathologies in adults or deformities during embryogenesis. It
has recently become clear that both steroids and retinoids play a major
role during animal development, serving as signaling molecules which influence
the processes that result in normal body shape and tissue differentiation.
Retinoids and steroids are known to modulate gene expression via binding
to nuclear receptors (NRs), proteins encoded by a highly conserved family
of genes that act as specific transcription factors. Receptor protein
interactions in the presence of the signaling ligand mediate their interaction
with DNA or associations with other regulatory proteins, in turn affecting
gene transcription. The molecular mechanisms through which specific
steroids and retinoids exert their physiological effects remain unclear.
We are investigating these mechanisms in an invertebrate model system-
the fiddler crab, Uca pugilator- in collaboration with P. M. Hopkins,
an endocrine physiologist in our department. Our research
is examining the type and distribution of signaling molecules present during
the growth cycle, and determining how these signaling molecules interact
with specific ecdysteroid receptor proteins to regulate gene expression.
We are particularly interested in this system due to the concerted growth
and developmental activities that may be under ecdysteroid and retinoid
influence during the crustacean adult life cycle; growth, reproduction,
and limb regeneration, a system amenable to experimental manipulation.
To initiate this study, we have isolated cDNA clones from the fiddler crab
representing homologs of the ecdysteroid (UpEcR) and retinoid-X
(UpRXR) classes of NR, which are known to form the functional ecdysteroid
receptor in insects. These clones have provided data on the sequence
of these molecules and their structural relationship to other members of
the nuclear receptor superfamily. The amount and type of receptor
that may be present in a given cell is difficult to measure directly.
Recombinant DNA techniques, however, can be used to develop molecular probes
that can mark the presence of specific receptors at the cellular level,
and microbes can be engineered to produce large amounts of receptor protein.
These methodologies are being employed to detect which cells contain receptors,
and by inference, identify particular cells that may be responding to steroid/retinoid
signaling. The recombinant proteins can also be used to examine the
physical characteristics of the receptors, such as their ability to bind
to specific signaling molecules and the DNA sequences of potential target
genes. Experiments with recombinant proteins are currently underway
to determine their DNA binding characteristics, and we are initiating production
of recombinant proteins in cell culture to produce sufficient amounts for
ligand-binding studies. |
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