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Abstract
Saccharomyces cerevisiae or Baker’s yeast is a
commonly used model organism for researchers
who study cell division and the genes that are
involved in its regulation. Several yeast cell
division genes have human versions with putative
roles in the formation of cancerous tumors. One of
these cell division genes, CDC7, is also involved in
meiotic division although its precise role in meiosis
is unknown. One way to help elucidate CDC7’s role
in yeast meiosis is to determine when Cdc7 protein
is made using western analysis. In order to
perform this analysis, antibodies are required that
will specifically recognize the Cdc7 protein. Since
none are yet available, yeast strains were
constructed that contain the CDC7 gene tagged
with a portion of the human myc gene. The tagged
CDC7 gene has the same phenotype as the
wild-type gene. Antibodies were then purchased
that recognize the human myc portion of the
Cdc7-myc protein. Experiments were done to
determine the appropriate antibody concentrations
necessary to detect the tagged Cdc7 protein from
mitotic cells on immunoblots. This information will
be used to perform a western analysis of protein
obtained from meiotic yeast samples to determine
when during meiosis the Cdc7 protein is expressed.
Introduction
One of the ways being investigated to
understand the spread of cancer is to determine
how proteins affect normal cell growth processes.
Saccharomyces cerevisiae or Baker’s yeast is
studied by thousands of researchers because yeast
contains cell division proteins that are similar to
those in human cells. One commonly studied yeast
cell growth protein is Cdc7. This protein kinase is
required for initiating DNA replication (S phase)
during the mitotic cell cycle, although it is not
understood how Cdc7 protein controls this
initiation in yeast.
Saccharomyces cerevisiae is a single-celled
eukaryote that can exist as a diploid or as a
haploid. Haploid yeast can be one of two forms:
“a” or “.” These two forms can mate with each
other to form a/ diploid cells. All of these cell
types, a, , and a/ diploids, can grow mitotically,
but only a/ diploids can undergo the cell division
process of meiosis.
Meiosis is a process similar but not identical to
the mitotic cell cycle. It is worthwhile to examine
the role of Cdc7 protein in meiosis to help
understand its role in the mitotic cell cycle and
possible cancer development. Currently, very little
is known about what Cdc7 does in meiosis
although it is known to be required for meiosis to
occur properly in yeast. One way to examine the
meiotic role of Cdc7 is to analyze Cdc7 protein
levels throughout meiosis. We constructed a strain
containing the CDC7 gene tagged with myc so that
Cdc7 protein can be detected by western analysis
using commercially available anti-myc antibodies.
Results
Two haploid temperature sensitive cdc7 mutant
yeast strains were separately transformed with a
plasmid containing the wild-type CDC7 gene with a
portion of the human myc gene fused to its 3’ end
(Figure 1). Transformants were selected on media
containing uracil. These transformants contained
the cdc7ts mutation, the URA3 gene that allows
them to grow and be selected on media lacking
uracil, and a second copy of CDC7 with the myc tag
(Figure 1). The transformants were grown on YPD
media which contains uracil, allowing the survival
of any cells that removed one of the copies of CDC7
and the URA3 gene by recombination (Figure 1).
If the crossover occurred to the left of cdc7ts as
drawn in Figure 1, the resulting “popout” would
contain the CDC7-myc gene, the desired genotype.
If recombination occurred to the right of cdc7ts as
depicted (Figure 1) the resulting “popout” would
contain the cdc7ts mutation and would be identical
to the original strain. This was not the desired
genotype. In either case, popouts would lose the
wild-type URA3 gene and would be unable to grow
on media lacking uracil. 5-FOA media was used
to select for the growth of these Ura- yeast popouts
because the metabolic poison 5-FOA kills Ura+
cells and thus allows only Ura- cells to grow.
To distinguish between the two possible
genotypes, the popouts were tested to determine if
they were cdc7ts or CDC7-myc by testing for
temperature sensitivity. The cells were replica
plated to rich YPD media and incubated at 22C or
30C. Popouts that are CDC7-myc, the desired
genotype, are not temperature sensitive and should
grow at both 22C (the permissive temperature)
and 30C (the restrictive temperature). Popouts
that are cdc7ts, the undesired genotype, should only
grow at the permissive temperature of 22C.
Table 1 shows that 8 out of 13 “a” popouts and 4
out of 4 “” popouts were no longer temperature
sensitive, indicating that they contained the desired
CDC7-myc construct.
“a” and “” popouts were crossed together to
form diploid strains. The haploid integrants, the
haploid popouts, and the diploid strains were
grown up in rich media and protein was isolated.
The protein was loaded onto a polyacrylamide gel,
subjected to electrophoresis, transferred to
nitrocellulose membrane, and analysed by
immunoblot (Figure 2A).
The diploid strains were then induced to
undergo meiosis by starvation. Samples were taken
after 0, 15, and 16 hours of meiosis. Protein was
isolated and immunoblotting was performed as
described above. The results are shown in Figure
2B.
cdc7ts
cdc7ts
URA3
CDC7-myc
cdc7ts
CDC7-myc
cdc7ts
CDC7-myc
Figure 1. Construction of yeast strains containing
CDC7 tagged with myc. A) A plasmid containing
the CDC7-myc construct was transformed into a
cdc7ts strain. This plasmid integrated into the yeast
chromosome by recombination, resulting in a
strain that had two copies of the CDC7 gene with
an intervening URA3 gene. B) Crossing over then
occurred spontaneously between the two copies of
CDC7 resulting in the loss of the URA3 gene and
one of the two CDC7 genes. If the crossover
occurred on the left side of cdc7ts, as depicted, then
the "popout" contains the desired CDC7-myc
construct.
Table 1. Results of testing "popouts" for
temperature sensitivity.1
“a” popouts YPD 22C
1
2
3
4
5
6
7
8
9
10
11
12
13
+
+
+
+
+
+
+
+
+
+
+
+
+
“” popouts YPD 22C
1
2
3
4
1
+
+
+
+
YPD 30C
+
+
+
+
+
+
o
o
o
o
+
+
o
YPD 30C
+
+
+
+
A"+" indicates growth; a "o" indicates no
growth.
1
6
7
2
7
4
5
8
1
6
3
2
3
4
5
8
Figure 2. Results of Cdc7 immunoblot.
A) Yeast strains were grown mitotically. Protein
was
isolated
and
subjected
to
SDS-polyacrylamide gel electrophoresis. After
transferring the protein to a nitrocellulose
membrane, an immunoblot was performed
using antibodies specific to the myc tag. Bands
on the X-ray film represent the locations of the
Cdc7-myc protein in each lane. Lane 1:
negative control; Lane 2: positive control;
Lane 3: haploid “a” integrant; Lane 4: haploid
“a” popout; Lane 5: haploid “” integrant;
Lane 6: haploid “” popout; Lane 7: diploid
myc-containing strain #1; Lane 8: diploid
myc-containing strain #2. The anti-myc
antibody was specific to the Cdc7-myc protein;
no other bands were observed on the X-ray
film.
B) Yeast strains were grown mitotically and then
were induced to undergo meiosis by
starvation. Protein was isolated and an
immunoblot was performed using antibodies
specific to the myc tag as described above.
Lane 1: negative control; Lane 2: positive
control; Lane 3: diploid #1 at 0 hrs.; Lane 4:
diploid #1 at 15 hrs.; Lane 5: diploid #1 at 16
hrs.; Lane 6: diploid #2 at 0 hrs.; Lane 7:
diploid #2 at 15 hrs.; Lane 8: diploid #2 at 16
hrs. The anti-myc antibody was specific to the
Cdc7-myc protein. A protease is likely to be
responsible for the extra bands observed in the
lanes containing meiotic proteins (lanes 4, 5, 7,
8).
Discussion
Two haploid yeast strains, an “a” and an “”,
were engineered to contain the CDC7 gene tagged
with a portion of the human myc gene to enable us
to detect Cdc7 protein in meiosis using
commercially available anti-myc antibodies. A
phenotypic assay confirmed the presence of this
construct in both haploids.
The “a” and “” CDC7-myc haploids were
mated together to construct an a/ diploid that
could undergo meiosis. This diploid was used to
develop a protocol for western analysis so that the
expression of the Cdc7 protein could be examined
throughout meiosis by immunoblotting.
Although meiotic Cdc7p is detectable by
immunoblotting, it seems that there is a protease in
the meiotic protein samples that is degrading the
Cdc7 protein (See Figure 2B). When this
experiment is repeated, protease inhibitors will be
added to alleviate the problem.
Future Work
 The immunoblot experiment shown in Figure 2B
will be repeated using protease inhibitors.
 Once the degradation problem is resolved a large
scale experiment will be set up in which samples
will be collected each hour for 12 hours followed
by a final time point at 24 hours. Protein will be
isolated and an immunoblot will be performed to
determine when during meiosis Cdc7 protein is
being produced.
Acknowledgements
We would like to thank Dr. Anne Galbraith for her
time and efforts in helping us with this research
project. This work was funded by a UW-L
Undergraduate Research Grant to Jacqueline
Bjornton and a UW-L Faculty Research Grant to
Dr. Anne Galbraith.