Previous studies have shown that the expression of E1A proteins
in
S. cerevisiae
inhibits cell growth in G1,
and that CR3 is partially responsible for this inhibition
(26; 73).
Confirmation of
these phenotypes is illustrated in
Fig. 1
and
Fig. 2.
Figure 1
Effect of E1A proteins on yeast cell growth.
Strain MSY596 was transformed with control vector
and E1A plasmids
and transformants were streaked on SGC plates to induce expression of E1A.
Growth was determined after 3 days of incubation at 28C.
The structures of the deletion mutants are illustrated in
Figure 3.
(A)
control vector;
(B)
E1A289;
(C)
E1A243;
(D)
dl1101;
(E)
dl1104;
(F)
dl1107
Figure 2
FACS analysis of yeast cells expressing E1A proteins.
Plasmid transformants of MSY596 were grown in liquid
culture in glycerol-ethanol medium and then induced with
2% galactose for 4 hours.
FACS analysis was performed as described previously
(Smith 1991).
(A)
control vector;
(B)
E1A289;
(C)
E1A243;
(D)
dl1101;
(E)
dl1104;
(F)
dl1107;
(G)
pMS424;
(H)
pMA424.82T (Gal4p-E1A1-82)
Briefly,
cells expressing either
E1A243
or
E1A289
under control of the
GAL1 promoter showed a pronounced inhibition of colony growth on galactose
relative to cells with the control vector
(Fig. 1A-C).
Following galactose induction,
both
E1A243
and
E1A289
resulted in an accumulation of cells with a G1 DNA content
as assayed by flow cytometry
(Fig. 2A-C).
A comparison of the growth phenotypes of cells expressing
E1A243 with those expressing E1A289
shows that CR3 is partially responsible for E1A function
(compare
Fig. 1B with 1C,
and
Fig. 2B with 2C).
However,
E1A243
is still a potent inhibitor of yeast cell growth
(Fig. 1C and 2C),
suggesting that the functional domains involved might coincide with
those critical for cellular gene activation,
repression, and transformation in mammalian cells.
Next:Mapping functional domains of E1A
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Copyright 1995 Stockton Press
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November 26, 1995 at 9:52 PM