exon selection regulated? Minigene constructs con- 
taining different arrangements of the a-tropo- 
myosin (a-TM) exons 2 and 3 have been used for in 
vivo and in vitro analyses. The mutual exclusivity is 
based on the peculiar anatomy of the intron be- 
tween these two exons. The branch point sequence 
is located very distantly from the acceptor site and 
too close to the donor site. This proximity creates 
steric hindrance between the splicing factor bound 
to the 5' splice site and the branch point that pre- 
vents a productive interaction. When this distance 
is increased, the mutually exclusive behavior is 
eliminated. These experiments have been explored 
to redefine the characteristics of the 3' splice site of 
the intron. The results indicate that the branch 
point of an intron is defined by a constrained se- 
quence and its close association with a poly- 
pyrimidine tract. The 3' splice site, on the contrary, 
does not require a polypyrimidine and is defined as 
the first AG downstream of the branch point. The 
choice between mutually exclusive splice sites, at 
least in the a-TM gene, is governed by the strength 
of the polypyrimidine tract. Swapped experiments 
in vivo and in vitro have demonstrated that it is 
possible to change the pattern of exon usage in a 
predictable manner. The behavior of different poly- 
pyrimidine tracts in splicing correlates with their af- 
finity for the splicing factor U2AF, as demonstrated 
by UV crosslinking experiments. 
III. Analysis of Genes Involved in the Regulation of 
the Contractile State. 
To address some of the unresolved aspects of ex- 
citation-contraction coupling, the laboratory has 
cloned from skeletal and cardiac muscle two of the 
main genes involved in the process, the a-subunit 
of the dihydropyridine-sensitive calcium channel 
and the calcium release channel of the sarcoplas- 
mic reticulum. Expression vectors with the wild- 
type and in vitro mutagenized sequences are being 
tested, in order to initiate a systematic analysis of 
structure-function relationships in these molecules. 
At the same time the kinetic properties of a cloned 
skeletal muscle potassium channel that is the ho- 
mologue of the Shaker channel in Drosophila 
melanogaster have been analyzed in mammalian 
cells and in frog oocytes. From the kinetic proper- 
ties a functional model of the channel that is exper- 
imentally testable has been developed. A systematic 
mutation of the S4 region of this channel is under 
way, to test the model and elucidate the basis for 
the voltage sensitivity of this molecule. These ex- 
periments are being carried out in collaboration 
with Dr. Peter Hess (Harvard Medical School). 
Dr. Nadal-Ginard is also Professor of Pediatrics 
and of Cellular and Molecular Physiology at Har- 
vard Medical School. 
PUBLICATIONS 
Books and Chapters of Books 
Breitbart, R., and Nadal-Ginard, B. 1988. Tissue specific alternative splicing of the troponin T multigene fam- 
ily. In Tissue Specific Gene Expression (Renkawitz, R., Ed.). Weinheim: VCH-Verlagsgesellschaft, pp 199- 
215. 
Mahdavi, V, Lompre, A.M., Chambers, A.R, and Nadal-Ginard, B. 1988. Regulation and structure of the a- and 
P-cardiac myosin heavy chain genes. In Pediatric Cardiology: Atrioventricular Septal Defects (Jimenez, 
M.Q., and Martinez, M.A., Eds.). Madrid: Ediciones Norma, pp 369-387. 
Articles 
Donoghue, M. , Ernst, H. , Wentworth, B., Nadal-Ginard, B. , and Rosenthal, N. 1988. A muscle-specific en- 
hancer is located at the 3' end of the myosin light-chain 1/3 locus. Genes Dev 2:1779-1790. 
Endo, T, and Nadal-Ginard, B. 1989. SV40 large T antigen induces reentry of terminally differentiated 
myotubes in the cell cycle. UCLA Symp Mol Cell Biol 93:95-104. 
Mahdavi, V, and Nadal-Ginard, B. 1989. Molecular basis of cardiac contractifity. Heart Failure 5:135-140. 
Smith, C.WJ. , Gallego, M.E., Andreadis, A. , Breitbart, R., Knaack, D. , and Nadal-Ginard, B. 1988. Alternative 
splicing of contractile protein minigene constructs is directed by cis and trans mechanisms. UCLA Symp 
Mol Cell Biol 67:265-277. 
Continued 
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