In contrast, the other two genes have a very re- 
stricted pattern of expression. One is expressed, at 
the mRNA and protein level, preferentially in car- 
diac cells and neurons. The other is expressed in 
skeletal muscle and cortical neurons. With the assay 
systems available, no functional differences have 
been detected among the different isoforms. Car- 
diac and smooth, as well as skeletal, muscles 
contain functionally saturating levels of MEF2- 
transactivating factors that are absent from the non- 
muscle cells. Moreover, MEF2 is induced in non- 
muscle cells by MyoD; however, MEF2 alone is 
insufficient to produce the full muscle phenotype. 
These results indicate that, although MEF2 is in- 
duced in skeletal muscle by the MyoD family of regu- 
lators, other lineage-determining pathways must 
lead to MEF2 expression in nonskeletal muscle tis- 
sue, where the MyoD family is not expressed. 
Whether basic helix-loop-helix (bHLH) or other 
factors induce the MEF2 genes in cardiac and 
smooth muscle, as well as neurons, the regulatory 
sequences of these genes will serve as powerful 
tools for the dissection of the lineage-dependent 
pathways in these cell types. 
Biochemistry of the Terminally 
Differentiated Phenotype 
The production and maintenance of the termi- 
nally differentiated phenotype of striated muscle 
cells involves the permanent withdrawal from the 
cell cycle and the induction of muscle-specific 
genes. A common biochemical explanation for these 
two processes and for the mutual exclusivity be- 
tween cell growth and differentiation has been lack- 
ing. Evidence recently obtained in Dr. Nadal- 
Ginard's laboratory indicates that central to these 
processes is the retinoblastoma gene product 
(pRB), which is required for the production and 
maintenance of the terminally differentiated pheno- 
type of muscle cells. Inactivation of pRB, either 
through phosphorylation, binding to oncogenes, or 
genetic alteration, inhibits myogenesis. Moreover, 
inactivation in terminally differentiated cells allows 
them to re-enter the cell cycle. pRB is required for 
the myogenic and trans-activation activities of 
MyoD. In addition, pRB is required for the cell 
growth inhibitory activity of MyoD. Both in vivo and 
in vitro, pRB binds directly to MyoD through the 
pocket and the bHLH domain, respectively. This di- 
rect binding induces a family of transcription factors 
that operate through the MEF2 DNA-binding site, 
present in most, if not all, muscle-specific genes, as 
indicated in the previous section. 
The fact that cell growth and differentiation are 
mutually exclusive in myogenic, as well as other 
terminally differentiated cell types, is a puzzle that 
until now has defied a molecular explanation. The 
dual role of pRB in cell cycle control and muscle- 
specific gene transcription explains a major aspect 
of this apparent paradox. Unphosphorylated pRB is 
required for myogenesis and muscle-specific gene 
expression through its interaction with MyoD; spe- 
cific phosphorylation of pRB is required for cell cy- 
cle progression. Thus, through its mutually exclu- 
sive interaction with cell cycle regulators and 
myogenic factors, pRB can function as a binary 
switch that determines whether the cell progresses 
through the cell cycle or commits to the terminally 
differentiated pathway. 
At the basis of the model described here are the 
multiple interactions available to each of the main 
players responsible for cell differentiation and 
tissue-specific gene expression. This striking fea- 
ture permits them to function as binary switches. In 
the presence of high concentrations of growth fac- 
tors, each of these regulators can further stimulate 
cell growth; under low-growth conditions, each can 
serve as a cell growth suppressor and play a role 
in the production of the terminally differentiated 
phenotype. By replacing MyoD for other lineage- 
determining genes, the model emerging from myo- 
genesis is likely to apply to other terminally differ- 
entiated systems. 
Alternative Splicing of Contractile 
Protein Genes 
The laboratory's mechanistic analysis of alterna- 
tive splicing has concentrated on the a-tropomyosin 
exons 2 and 3- These exons are mutually exclusive: 
exon 3 is found in the mRNA of most cells (the de- 
fault pathway) ; exon 2 is found only in the mRNA of 
smooth muscle (the regulated pathway). The pri- 
mary determinant for the default pathway is the 
strength of the branch point/polypyrimidine tract 
upstream of these two exons. Dr. Nadal-Ginard and 
his colleagues previously demonstrated that the 
strength of a given exon in cis-competition splicing 
assays is directly correlated with the ability of the 
polypyrimidine tract-binding protein (PTB), char- 
acterized in this laboratory and in Dr. Phillip Sharp's 
laboratory (Massachusetts Institute of Technology) , 
to be crosslinked to the polypyrimidine tract of the 
exon. The interaction of PTB with the pre-mRNA has 
been exploited to investigate the very early step in 
spliceosome assembly and to determine when 
splice site selection and commitment to a given 
splicing pattern occurs. These studies have revealed 
that Ul snRNP associates with the 3' end of the in- 
tron in the earliest step of spliceosome formation in 
an ATP-independent manner. Since it has been 
GENETICS 231 
