neuropeptides that are expressed differentially and 
in discrete regions in the nervous system. 
These independently regulated genes offer an ex- 
cellent model system in which to explore the deter- 
minants of tissue-specific gene expression. Dr. 
Amara and her colleagues have examined the reg- 
ulatory role of 5' upstream sequences of a- and P- 
CGRP genes by analyzing the expression of gene 
constructs introduced into cells after transient DNA 
transfection. Promoter regions have been fused 
to reporter genes. Constructs containing a- and 
P-CGRP 5 '-flanking regions direct the expression of 
chloramphenicol acetyltransferase (CAT) activity in 
several cell lines derived from neural, endocrine, 
and nonneural tissues. Constitutive a- and (B-CGRP 
promoter-driven expression is low relative to ex- 
pression from a strong viral promoter (SV40) but 
can be induced severalfold after exposure of cells 
to various hormonal agents. Deletion mapping of 
the upstream promoters is being utilized to delin- 
eate further the functional regions that mediate ob- 
served hormonal effects. 
III. Regulated Pre-mRNA Processing. 
Alternative pre-mRNA processing is an important 
mechanism for regulating the diversity of gene 
products. A wide array of gene products are en- 
coded within complex transcription units that gen- 
erate multiple mature mRNAs through the selective 
use of multiple start sites, spUce sites, and/or poly- 
adenylation sites. Whether the crucial regulatory 
events of the calcitonin/a-CGRP gene involve the 
selective use of polyadenylation sites, the selective 
use of 3' splice sites, or both, is controversial. It is 
equally unclear whether a specific trans-acting fac- 
tor regulates the CGRP splice choice, with calcito- 
nin produced by a default pathway, or vice versa, or 
whether each pathway requires specific factors. 
Having established the baseline pattern of mRNAs 
produced in several cell lines. Dr. Amara and her 
colleagues have also introduced several mutated 
and hybrid genes into cultured cells to identify se- 
quences critical for splice regulation. 
PUBLICATIONS 
The mechanism of pre-mRNA processing reg- 
ulation can be considered from two perspectives, 
each of which suggests an experimental approach. 
One determinant of a particular processing choice 
clearly resides within the primary RNA transcript, 
either as a specific sequence or secondary struc- 
tural feature. The other determinant resides in 
the enzymatic machinery that recognizes these 
specific structural features and targets the tran- 
script to a particular processing pathway. For regu- 
lated processing events, tissue-specific differences 
in the basic processing machinery must be pos- 
tulated. 
Although transfection and mutagenesis studies 
should provide insight into the cis-acting sequences 
that influence alternative processing, a major goal 
is to learn more about the trans-acting factors that 
influence these events. Therefore, studies have 
been directed toward establishing a convenient 
assay system in which to test effects of exogenously 
added factors on the splicing patterns. One ap- 
proach has been to microinject the calcitonin/a- 
CGRP gene into the Xenopus laevis oocyte nucleus 
and analyze the RNA products that result. The gene 
constructs used for these experiments contain the 
calcitonin/a-CGRP gene under the control of its 
own promoter or, alternatively, fused to a viral pro- 
moter known to work well in the oocyte. The pat- 
tern of basal expression of the calcitonin/a-CGRP 
gene suggests that the oocyte nucleus transcribes 
the gene well but lacks the machinery necessary to 
make the regulated processing steps. 
Studies on tissue-specific snRNPs, snRNAs, and 
putative factors involving alternative pre-mRNA 
processing continue as a collaborative project with 
Dr. Michael R. Lerner (HHMI, Yale University 
School of Medicine). Co-injection experiments 
using exogenous nuclei, nuclear extracts, enriched 
snRNP preparations, or snRNAs are under way to 
identify specific factors that influence processing 
events. 
Dr. Amara is also Assistant Professor of Molecular 
Neurobiology at Yale University School of Medicine. 
Articles 
Blakely, R.D., Robinson, M.B., and Amara, S.G. 1988. Expression of neurotransmitter transport from rat brain 
mRNA in Xenopus laevis oocytes. Proc Natl Acad Sci USA 85:9846-9850. 
McAllister, G., Roby-Shemkovitz, A., Amara, S.G., and Lerner, M.R. 1989- cDNA sequence of the rat U snRNP- 
associated protein N: description of a potential Sm epitope. EMBO / 8: 1177-1181. 
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