Project: Research project

Project Details


Mechanisms controlling gene expression in eukaryotic cells are complex,
encompassing transcriptional and post-transcriptional components. Due to
inherent difficulties in their study, post-transcriptional mechanims have
received relatively less attention; in particular, RNA transport has been
studied as the appearance of rapidly-labeled RNA in the supernate after
incubation of nuclei in vitro. Such assays would be more firmly accepted
if the transported RNA was better characterized. It is the intent of this
proposal to test the fidelity of carefully selected in vitro system by
examining the behavior of specific sequences. Using perturbations which
result in great differences in mRNA formation and abundance in vivo,
metabolism of an acute phase reactant sequence (alpha1-acid glycoprotein),
a hormonally-responsible globulin sequence (alpha2Mu-globulin), and marker
sequences for mature (albumin) and primitive (Alpha-fetoprotein) liver
cells will be examined with an in vitro transport system. An additional
model will employ an analbuminemic rat strain, which transcribes albumin
sequences into nuclear RNA but restricts them to the nucleus. Transported
and nucleus-restricted RNA will be isolated and in vitro translation
products will be examined. RNA will be separated by size
electrophoretically, "Northern blotted" and hybridized with specific
P32-cDNA probes, and autoradiographs will be developed. Mixing experiments
will document and quantitate cytoplasmic contamination of nuclear and
transported preparation. These experiments will determine whether in vitro
transport assays can support processing/transport of correctly-sized
functional mRNA, with modulation paralleling in vivo circumstances.
Providing specificity can be established (as preliminary results indicate),
use of in vitro transport assays should contribute valuably to
understanding controls of gene expression, especially the altered
post-transcriptional RNA transport associated with carcinogenesis. If
specificity cannot be established, the considerable effort and funding
devoted to their use in studies of "in vivo specificity" should be
Effective start/end date12/31/891/31/02