In mammalian cells the N-terminal region and CR1 can function
together to either activate or repress transcription.
We have previously shown that the N-terminal region and CR1,
but not CR2,
function together to activate transcription of the c-fos
proto-oncogene
(22; 23),
and others have shown that these domains act
to repress transcription of a variety of genes
(1; 2; 33; 37; 47; 68).
The results reported here implicating both the N-terminal region and CR1,
but not CR2,
in inhibition of yeast cell growth
suggest a functional similarity with the mammalian system.
To pursue the function of these two domains in yeast,
we asked whether the N-terminal region and CR1 can function
independently to inhibit yeast cell growth.
Residues 1-82 of E1A were cloned under the control of the
GAL1 promoter,
and induced in cells by growth on galactose as before.
Unlike full length E1A,
this construct failed to inhibit growth on galactose plates.
However,
we were also unable to detect any expression of this protein
by Western blot analysis,
suggesting that the lack of phenotype was due to a failure to
express the polypeptide.
To stabilize the E1A polypeptide we engineered a hybrid gene in
which residues 1-82 were fused with the DNA binding domain of
the yeast transcription factor Gal4p
by inserting the E1A sequences into plasmid pMA424
(12).
The resulting construct is a high copy number plasmid
in which the fusion gene is expressed from the constitutive
ADH1 promoter.
Expression of the
Gal4p-E1A1-82
polypeptide from this plasmid
strongly inhibited cell growth in all strains tested.
In the yeast host MSY596,
transformants gave small poorly growing colonies,
while in several other laboratory strains
we failed to obtain any transformants at all with this plasmid
(Fig. 6A,B).
Figure 6
Cells that are
net1-1
are resistant to expression of Gal4p-E1A1-82
but not Gal4p-VP16.
MSY596 (wild type)
or MSY826 (net1-1)
cells were grown to exponential growth
and transformed with either pMA424,
pMA424.82T (Gal4p-E1A<1-892>),
or PadhGV16 (Gal4p-VP16).
Transformations were plated and examined for colony growth.
(A)
MSY596 with pMA424;
(B)
MSY596 with pMA424.82T;
(C)
MSY596 with PadhGV16;
(D)
MSY826 (net1-1) with pMA424.82T;
(E)
MSY826 (net1-1) with PadhGV16
Flow cytometric analysis of MSY596 transformants
showed that the growth inhibition
by the fusion protein was predominantly in G1
(Fig. 2G,H).
The parent vector pMA424 transformed at high efficiency
and supported normal growth and cell cycle progression.
These results suggested that the first 82 residues of E1A
could function to inhibit cell growth in G1
independent of the other domains of the E1A proteins.
Next:Yeast mutants resistant to E1A
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Previous:Nuclear localization
Copyright 1995 Stockton Press
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November 26, 1995 at 9:52 PM