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A GENETIC SCREEN FOR MCM10: INTERACTIONS BETWEEN MCM10 AND DEFICIENCY REGIONS ON
THE 3RD CHROMOSOME OF DROSOPHILA MELANOGASTER
by
Bryan J. Anstead
A Senior Honors Project Presented to the
Honors College
East Carolina University
In Partial Fulfillment of the
Requirements for
Graduation with Honors
by
Bryan J. Anstead
Greenville, NC
May 2015
Approved by:
Dr. Tim Christensen
Department of Biology, Thomas Harriot College of Arts and Sciences
ABSTRACT - Mis-regulation of the Mcm10 gene has been linked with multiple
forms of cancer, including many forms of breast cancer (Thu, Y. 2014). Despite this, the role
of Mcm10 in cancer is not yet fully known. One way to study a gene is to see the effects
when it is no longer functioning. When eukaryotic organisms are homozygous deficient for
Mcm10 they are still able to function despite the complete loss of the Mcm10 protein
(Christensen, T. 2003). This suggests that there are other genes that interact with Mcm10
which are able to make up for its’ loss, allowing the organism to continue to function. The
hypothesis is that by studying the interactions between Mcm10 and other genes, Mcm10’s
role, and its carcinogenic effects, can be more fully understood. Genetic screening will aid in
this study of Mcm10 and its interacting genes, as well as their role in cancer. We propose an
enhancer/suppressor screen of the third chromosome of Drosophila melanogaster, spanning
roughly 7,619 genes, to determine Mcm10’s mechanisms of interaction.
2
Acknowledgments
I would like to thank the people and organizations that made this study possible:
Ariana Rebaza, Jerri Strickland, Kim Valle, Brenda Kennedy, the Department of Biology,
East Carolina University, and Dr. Elizabeth Ables. I am especially grateful to my project
advisor, Dr. Tim Christensen, for his knowledge and guidance. Funding was received from
East Carolina University Undergraduate Research and Creativity Award.
3
Table of Contents
Introduction
7
Methods & Materials
15
Fly Husbandry
15
Fly Collection and Cross Setting
15
Deficiency Lines Used
16
Data Analysis and Gene Identification
17
Results & Discussion
18
Df(3R)BSC874: Mcm10 & Cul5
20
Df(3L)BSC839 & Df(3R)ED6255: Mcm10 & pol-α
22
Df(3L)ED4789: Mcm10 & Reptin
23
Df(3R)ED5622: Mcm10 and Orc2
23
Df(3L)BSC816
24
Df(3L)ED201
25
Df(3R)ED7665
26
Df(3R)ED5100
26
Df(3R)ED5516
26
Df(3R)BSC568
27
Df(3R)BSC748
27
Df(3L)BSC12
27
Df(3L)ED4543
28
Df(3R)ED10555
28
Df(3R)ED10845
29
Df(3R)ED5330
29
Conclusion
31
Literature cited
32
4
List of Tables
Table 1: Determination of an Interaction
14
Table 2: Stocks ordered from the Bloomington Drosophila Stock Center
16
Table 3: Each deficiency line outside of the standard deviation
19
5
List of Figures
Figure 1: Example Deficiency Region
11
Figure 2: Schematic representation of crosses
13
Figure 3: Graphical Representation of each Wt/(CyO+Sb) Ratios calculated
18
Figure 4: Graphical representation of the results from the two crosses
performed between the single Cul5 mutation and Mcm10148.
21
Figure 5: Graphical representation of the progeny from
Df(3R)ED5330 x Mcm10148.
30
6
Introduction
Mcm10 is a protein that may have separate roles in DNA replication, endoreplication,
the cell cycle, DNA damage repair, origin firing, and stem cell maintenance (Apger, J 2010;
Thu, Y. 2013; Ricke, R. 2004). Despite this, the true function of Mcm10, in replication and
as a whole, remains elusive. Numerous studies on Mcm10 in single-celled organisms have
been performed, most notably in Saccharomyces cerevisiae. These studies provide much
information on Mcm10 as a whole, as it is a highly conserved gene, having a similar
appearance and likely a similar function throughout many organisms (Johnson, E. 2003).
Mcm10 is a very abundant protein, with approximately 40,000 copies in each haploid cell of
Saccharomyces cerevisiae, and is mainly localized in the chromatin or nuclear matrix during
the phases of the cell cycle (Kawasaki, Y. 2000). Mcm10 has been shown to associate with
the origin replication complex; Mcm10 is required during origin activations, as well as
during the disassembly of the pre-replicative complex. This is shown through the Mcm10
lesion causing drastic reduction in initiation at DNA replication origins (Merchant, A. 1997).
In the same study, the Mcm10 lesion caused pausing at replication forks during elongation,
suggesting a role for Mcm10 in the elongation step of DNA replication (Merchant, A. 1997).
Mcm10 has also been shown to interact genetically with initiation and elongation factors
Cdc45, DNA polymerase δ and ε (Kawasaki, Y. 2000). These studies present a strong
argument for Mcm10 having a role in the initiation and elongation of DNA replication, at
least in Saccharomyces cerevisiae.
Furthering our knowledge on Mcm10 in yeast, Mcm10 protein has been demonstrated
to associate and co-operate with other members of the Mcm family during the initiation of
DNA synthesis, and may function as a replication-licensing factor (Kawasaki, Y. 2000).
7
Building on this role in replication, Mcm10 mediates the loading of Mcm2-7 complex onto
replication origins (Homesley, L. 2000). Finally, other studies have been done determining
that accurate genome replication requires Mcm10, which interacts with the Mcm2-7 helicase,
DNA polymerase-α, the replication clamp, and the lagging-strand polymerase (Alver, Robert
C. 2014). Despite being a highly conserved protein, some evidence has arisen demonstrating
that not all aspects of Mcm10 are translated to higher organisms. Due to this, some of the
information gathered from these previous studies in yeast cells may not represent Mcm10 in
multicellular eukaryotes. For example, yeast Mcm10 binds to chromatin during the entire cell
cycle, with constant expression levels (Homesley, L. 2000; Kawasaki, Y. 2000). However,
human Mcm10 protein levels have been demonstrated to fluctuate during the cell cycle,
decreasing in late M/G1 phase, and bind to chromatin specifically in S phase (Izumi, M.
2001).
The study performed by Izumi was performed in human cell tissues. Like in
Saccharomyces cerevisiae, many studies have been performed on single-cell tissue cultures,
expanding our picture of what Mcm10 does. Through culture studies, Mcm10 was found in
the same genetic screen as the minichromosome maintenance family, which includes Mcm2Mcm7, which are all highly conserved throughout eukaryotes and are essential for DNA
replication (Johnson, E. 2003). Phosphorylation may be essential for the dissociation of
Mcm10 from chromatin, potentially by cdc2 kinase, although this requires clarification
(Izumi, M. 2001). Since Mcm10 deficiency is known to cause replication stress, and due to
its role in genome duplication and mis-regulation, Mcm10 may facilitate cancer
development. Through studies on cancer tissue samples, there has been shown to be a
relationship between Mcm10 mutations and multiple forms of cancer. Molecular studies,
8
mainly using Mcm2 and Mcm5, suggest that proliferative malignant cells, precancerous cells,
and potential cancer reoccurrence are marked by increased Mcm levels, especially in cervical
and lung cancers, as well as meningioma (Williams, G. 1998; Tan, D. 2001; Hunt, D. 2002).
Mcm10 could also play a role in this, due to its relationship with the Mcm complex. Mcm10
has been shown to be overexpressed in cervical cancer, showing increased frequency of
overexpression as tumor stages advanced, but this expression profile for Mcm10 remains
poorly understood (Das, M. 2013). Finally, Mcm10 overexpression is associated with WHO
(World Health Organization) tumor grade in glioma samples, one of the most common
malignant tumors of the central nervous system (Hua, C. 2014).
Extensive research has been performed on Mcm10 through these single-cell models.
However, little has been done in actual multicellular eukaryotic organisms. One in vitro
study, performed on Xenopus egg extracts, demonstrated MCM proteins become less tightly
associated with chromatin following entry of cells into quiescence, making Mcms effective
markers of proliferation (Madine, M. 2000). However, this study focused on all of the Mcm
proteins, rather than Mcm10 specifically. This serves to represent the deficiency in our
knowledge of Mcm10 in multicellular eukaryotes, despite the relationship with cancer in
human tissue cells, as well as its’ role in essential cell processes such as replication. To
reduce this gap in knowledge, we propose an enhancer/suppressor screen. Since organism
homozygous deficient in Mcm10 are still able to function, albeit imperfectly, there are likely
proteins that interact with Mcm10. The proposed genetic screen will serve to reveal and
determine the mechanisms between Mcm10 and the interacting genes.
Through the study of the interactions between Mcm10 with other genes, the function
of Mcm10 will be made clearer. This will be done through the performance of an
9
enhancer/suppressor screen for Mcm10 mutants in Drosophila melanogaster. A genetic
screen will be useful in further studies on Mcm10, allowing the mutation to be more
prominent. It will also shed light on possible non-essential processes Mcm10 may have a role
in. A genetic enhancer screen identifies mutations that increase a phenotype of interest in a
mutant of a different gene. The phenotype of this double mutant, one with both the enhancer
and original background mutation, is more noticeable than either of the single mutant
phenotypes. An enhancer screen can identify genes that redundantly with each other; a
suppressor screen determines genes that subdue the mutant phenotype caused by the original
mutation of interest. By performing enhancer and suppressor screens, a variety of genes can
be characterized to have an interaction with Mcm10. The screens will be performed using
classical genetic approaches.
Drosophila melanogaster, due to their ease of care, quick generation time, and
accessibility as a multicellular eukaryotic model organism, will be used to perform the
screen. This model organism was chosen in part due to its unique balancer chromosomes.
Balancers were created for all of the major chromosomes, and facilitate genetic analysis by:
suppressing recombination with their homologs; their presence being signaled by dominant
markers affecting adult and larval morphology; and having recessive mutations that cause
lethality or reduced fertility in heterozygotes (Casso, D. 1999). These properties allow for the
maintenance and stability of populations of mutants as balanced heterozygotes.
A genetic screen can be performed multiple ways. One is through the candidate-gene
approach. This method singles out individual genes that are predicted from the literature to
interact with a given gene. However, this method is very inefficient to perform a screen on a
scale as large as the proposed screen, as each individual gene out of the 7,619 would have to
10
be studied one at a time. Additionally, this method does not emphasize the discovery of novel
mechanisms. Using this method, only genes with some bias towards having an interaction are
likely to be studied if the project does not screen the entire chromosome. Another method is
to screen the entire chromosome by subdividing it into large deficiency regions. This will
dramatically increase the efficiency of the screen, as any region with an interesting result can
be further separated into the individual genes for further study. An example of this method is
shown in Figure 1. The red boxes are the possible deficiency regions for that span of the
chromosome, with the blue boxes representing the genes contained within that region.
Figure 1: Example Deficiency Region (Flybase, 2014). The red bars are different deficiency
regions, denoting the genes that are removed in that region. The blue pentagons represent a single gene. A
deficiency region could have over fifty genes in it, or as little as ten.
11
Our screen will be completed by crossing virgin females with a mutation in Mcm10,
located on the 2nd chromosome, with males having deficiency regions located on the 3rd
chromosome, which span multiple genes. To measure the results, two genetic markers will be
attached to the mutants with deficiencies: curly wings (compared to the wild-type straight
wings), located on the second chromosome, and stubble-hair (compared to the bristles found
in wild-type), located on the third chromosome. By studying the phenotypic ratio of progeny
from each cross, it will be able to determine if an interaction is present. Normally, if there is
no interaction, the four possible phenotypic ratios of the progeny will be equal: 1 wild-type,
containing both mutations, to 1 curly-winged, possessing only the deficiency region of
interest, to 1 stubble, having only the Mcm10 mutation, to 1 curly-stubble, lacking either of
the mutations. For a possible enhancer, the ratio will be similar to a 1:2:2:2 relationship, with
the amount of wild-type progeny, which contain both of the mutations, being much lower
than the other phenotypes. Conversely, the ratio for suppression would appear as a 2:1:1:1
relationship, with the wild-type progeny proliferating much more than either of the offspring
possessing the single mutations. If the number of offspring which contain deficiencies in both
mcm10 and the target region, shown as wild-type, are significantly lower than the number of
offspring with a deficiency in only the Mcm10 gene, shown through stubble, or the target
region, evidenced by curly wings, there is likely an interaction between Mcm10 and one of
the multiple genes in that deficiency range.
12
Figure 2. Schematic representation of crosses. This figure shows the crosses conducted. 30
Virgin female Drosophila melanogaster with the mcm10 mutant and curly-wing phenotypic marker were
crossed with 30 males that had the deficiency region on the third chromosome and the stubble-hair phenotypic
marker. All of the possible genotypes of the progeny is displayed, and the amount of each is counted and
analyzed to determine if there is any irregularities that could signify an interaction between Mcm10 and that
deficiency region. (Drosophila image source: Wikimedia Commons)
This is calculated through the expression
would be:
Genotypically, this calculation
An enhancer is characterized by a ratio much lower
than .50, ideally near .25; a suppressor is characterized by a ratio much higher than .50,
ideally near 1 based on the hypothetical relationships stated previously, as shown in Table 1.
13
No Interaction:
Wt/(CyO+Sb) Ratio = 0.5
Suppression:
Wt/(CyO+Sb) Ratio > 0.5
Enhancer:
Wt/(CyO+Sb) Ratio < 0.5
Table 1: Determination of an Interaction. If the ratio of Wild-type progeny to Curly and Stubble
progeny is around 0.5, an interaction is unlikely to be present. If the ratio is greater than 0.5, there is a
suppression interaction. If the ratio is less than 0.5, there is likely a gene within the deficiency region interacting
redundantly with Mcm10.
These ratios will determine whether an enhancer or suppressor gene is present. The
function of this gene will explain another role of Mcm10, as the two must work cooperatively
in order to exhibit enhancer or suppressor effects.
14
Methods
Fly Husbandry. Fly stocks were obtained from the Bloomington Drosophila Stock Center,
with each stock number listed in Table 2 below. Each stock was crossed with 175
Tm3,Sb/Tm6b,Tb,Dr mutants multiple times to add the stubble balancer. Virgin females of
the stocks lacking the stubble phenotypes were collected and crossed with the 175
Tm3,Sb/Tm6b,Tb,Dr mutants. After waiting ten days, the parents were dumped, and any
larvae that appeared to be tubby were squashed. All progeny with the stubble phenotype were
collected and stored in a vial together. The successive generations were monitored for the
stubble phenotype, and any stock still lacking the phenotype were re-crossed with the 175
Tm3,Sb/Tm6b,Tb,Dr mutants until the genotypes were cleaned of unwanted mutations.
Mcm10148 mutants were collected and maintained at room temperature.
Fly Collection. Female virgin Mcm10148 mutants were collected daily every six to eight
hours, to ensure virginity. The virgin females were stored five to a vial and monitored to
ensure no larvae were produced, preventing contamination.
Setting Crosses. Approximately thirty males of each stock number listed in Table 2 below
were collected and placed into a fresh bottle with the same amount of virgin female
Mcm10148 mutants. After a ten-day waiting period, the parents are dumped out of the bottle
and disposed of. The crosses were then scored every day over ten days, beginning the day
after the parents were dumped, and sorted into their respective phenotypes. After ten days,
the bottles were thrown away, to ensure only the F1 generation was scored.
15
Df(3L)ED4
293
8058
Df(3R)ED
5622
8959
Df(3L)ED4
789
8084
Df(3R)ED5177
8103
9215
Df(3L)ED4475
8069
Df(3R)ED5815
9208
Df(3R)ED10845
9487
Df(3R)ED6255
9210
Df(3R)X3F
2352
Df(3R)BSC874
29997
Df(3R)ED
6096
8684
Df(3R)ED
7665
8685
Df(3L)ED2
01
8047
Sxc bw
sp/SM5
3058
Df(3R)ED
6232
8105
P{RS3}Ca
m[UM8064-3]
8064
P{RS5}Dia
p15-HA-2788
2788
175
Df(3L)Exel9001
7924
Df(3L)BSC388/
TM6C
24412
Df(3R)BSC619/
TM6C
25694
Df(3L)BSC815
27576
Df(3R)Exel9029
7951
Df(3R)BSC819
27580
Df(3R)ED5938
24139
Df(3L)BSC673
26525
Df(3L)BSC816
27577
Df(3R)ED10639
9481
Df(3R)ED10555
23714
Df(3L)ED4543
8073
Df(3L)BSC220/
TM6C
9697
P{PcT:Avic/GFPEGFP}3
9593
Df(3L)BSC223/
TM6C
9700
Df(3L)BSC389/
TM6C
24413
Df(3L)BSC371/
TM6C
24395
Bq: klarmarb-CD4
st
25097
Df(3R)BSC738/
TM6C
26836
Df(3R)BSC621/
TM6C
25696
Df(3R)BSC568
25126
Df(3L)1-16
/TM6B
7002
P{Or67dGAL4.F}57.1
9997
Df(3R)Exel6270
7737
Df(3R)
Exel67
6
7743
Df(3R)BSC141/
TM6B
9501
Df(3R)Exel6182
7661
Df(3R)Exel6264
7731
Df(3L)ED4858
8088
Df(3L)BSC671
26523
Df(3R)BSC137/T
M6B
9497
Df(3R)ED5516
8968
Df(3R)ED5705
9152
Adhfn23 pr cn
l(2)46Ck26-19
9877
Df(3L)BSC839
27917
Df(3L)ED5017
8102
E l(3)CHf/DC10
9667
Df(3R)ED5100
9226
Df(3R)ED5339
9204
Df(3R)ED6346
24142
Df(3R)BSC748
26846
Df(3R)BSC547/
TM6C
25075
Df(3L)BSC
12
6457
Df(3R)BSC43
7413
Df(3L)BSC23
6755
Opt19926.3/SM1
3447
Df(3L)Aprt32/TM6
5411
Table 2: Stocks ordered from the Bloomington Drosophila Stock Center. The bolded items
are the specific deficiency names, and the number below it is the stock number. Each stock number represents a
different section mutated on the third chromosome of Drosophila melanogaster.
16
Df(3L)Exel6112
7591
Df(3L)ED2
08
8059
Df(3R)ED5
330
9077
Df(1)sd72b/
FM7c
3347
Data Analysis. The total amount of each phenotype that eclosed was calculated and used to
determine the individual ratios of wild-type to curly and stubble for the progeny of each
cross. These ratios were compared to the average of all of the stocks to determine the stocks
that greatly differed from the “normal” ratio through graphing with excel.
Identification of Genes. Once deficiency regions of interest are identified, the stock number
of that range is entered into Flybase in order to generate the Gbrowser displaying the genes
specific to that region. Each gene is then sorted through, and genes of interest are identified
by their molecular function and biological process performed. Genes associated with
functions such as: DNA binding, chromatin formation, DNA replication, primase, or
polymerase activity were chosen as a possible candidate for the enhancer or suppressor
effect, as well as interacting with Mcm10.
17
Results & Discussion
The final results of each cross are shown in Figure 3 below. The shaded region
indicates the range where the results of each cross was considered “normal,” and any result
outside of this was considered to have an candidate interaction. The range was determined
through the calculation of standard deviation of every result using Excel. The deficiency lines
that showed an enhancer or suppression effect are represented in Table 3, along with
deficiency lines that are near the edge of the standard deviation range. The cumulative
average eclosion day and associated ratio was also calculated, but no abnormal results were
found (Data not shown).
Figure 3. Graphical representation of each Wt/(CyO+Sb) Ratios calculated. The graph
shows the Wt/CyO+Sb ratios from all of the defiency lines crossed with Mcm10. The lines represent the range
of standard deviation in relation to the total data set, which is 0.13327. The average ratio for all of the results
was 0.47531. Any stock with a result that was outside of the standard deviation in relation to the overall data set
was determined to likely have a gene within it that interacts with Mcm10 in some fashion.
18
Enhancer
Suppression
Possible Interactions
Deficiency Line
& Stock
Number
Deficiency Line
& Stock
Ratio
Number
Deficiency Line &
Stock Number
Ratio
Ratio
Df(3L)ED201
(8047)
0.000
Df(3L)BSC816
(27577)
0.616
Df(3R)ED6255
(9210)
0.36
Df(3R)ED7665
(8685)
0.2838
Df(3R)ED5516
(8968)
0.629
Df(3L)ED4543
(8073)
0.372
Df(3R)ED5100
(9226)
0.2990
Df(3R)BSC568
(25126)
0.638
Df(3R)ED10555
(23714)
0.375
Df(3L)BSC839
(27917)
0.3030
Df(3R)BSC748
(26846)
0.652
Df(3R)ED10845
(9487)
0.378
Df(3R)ED5622
(8959)
0.663
Df(3R)ED5330
(9077)
0.387
Df(3L)ED4789
(8084)
0.733
Df(3R)BSC874
(29997)
0.562
Df(3L)BSC12
0.653
(6457)
Table 3: Each deficiency line outside of the standard deviation. The table displays the stock
number and Wt/CyO+Sb ratio for each result outside of the range of the standard deviation. The effect on
Mcm10 is shown at the top. Stocks that displayed abnormal results that had genes likely to interact with Mcm10
within the deficiency region are shown in the last column.
19
Df(3R)BSC874: Mcm10 and Cul5.
The cross with Df(3R)BSC874 (Stock #29997) initially showed a suppression effect.
Upon further trials, the results fluctuated from within and out of the standard deviation range.
Looking into the genes located in this deficiency line, Cul5 was identified, a gene in the
cullins family. The cullins gene family confer substrate specificity to E3-ligases, which are
involved in ubiquitin-mediated protein degradation or modification (Ayyub, C. 2011).
Similar to Mcm10, Cul-5’s role is not well understood. Multiple studies done have linked
Cul-5 to cellular proliferation and gene expression. Cul-5 mutants have significantly lower
cellular proliferation rates and inhibit cell growth (Dort, C. 2003). Mcm10 can be diubiquitinated, which is cell cycle regulated, appearing in late G1 and throughout S phase
(Das-Bradoo, S. 2006). Mcm10 must be di-ubiquitinated to bind to PCNA, and the diubiquitinated form of Mcm10 is associated with chromatin and DNA replication (DasBradoo, S. 2006). As Mcm10 must be ubiquitinated to perform all of its functions, the
ubiquitination could be done using Cul5, which would show either an enhancement or
suppression effect in the deficiency crosses performed and could account for the results seen
in Df(3R)BSC874.
An isolated Cul5 mutant, Cul521463, was crossed with Mcm10148. This was done
twice, with the total number of each phenotype shown below in Figure 4. The cross had a
final
ratio of 0.632, meaning that there is likely some suppression effect occurring
from the loss of both Cul521463 and Mcm10148, although a more in-depth study will need to be
conducted to explain this effect.
20
Figure 4. Graphical representation of the results from the two crosses performed
between the single Cul5 mutation and Mcm10148. The amount of Wt, or
higher than either of the two single mutations, indicated by Cyo and Sb, which gives a
is
ratio of 0.632,
indicating a suppression effect on the mutants.
The cul-5 homolog gene used by Dort, VACM-1, inhibits cell growth through a
mechanism involving MAPK and p53 signaling pathways. In Drosophila, the p53 gene is
located at 3R: 23,049,0657…23,054,082, which is contained in the deficiency line
Df(3R)ED6096 (Stock #8684) (Flybase.org). When crossed with Mcm10148, the
Ratio
was 0.555, within the range of standard deviation. However, the deficiency line was crossed
with only 15 males and 15 Mcm10148 virgin females, rather than the standard 25-30 of each,
and so the cross should be performed again to verify the results obtained. The MAPK genes,
P-38a and P-38b are located on separate chromosomes, with P-38b on 2L and P-38a on 3R.
However, the deficiency region contained P-38a was not crossed in this experiment, and
should be performed in further studies. VACM-1’s function is known to be regulated by
protein kinase A and protein kinase C. Exploring this in relation to Mcm10, it would be
21
relevant to check for an interaction between Mcm10 and these two proteins. Protein kinase C
was crossed in Df(3R)BSC874, stock 29997, which also contained Cullin-5. The protein
kinase A family has three genes located on the third chromosome of Drosophila
melanogaster, Pka-R1, Pka-C3, and Pka-C2, although we did not have the ability to cross
deficiency regions containing these genes (Flybase.org).
Df(3L)BSC839 & , Df(3R)ED6255: Mcm10 and pol-α
Df(3L)BSC839(Stock #27917) exhibited a enhancement effect, having a
ratio of 0.303. Upon examining the genes contained in this deficiency region, DNA
polymerase α 60 kD was identified; as mentioned before Mcm10 is known to interact with
DNA polymerase α. DNApol-α 60kD has a role in DNA primase activity, as well as the
synthesis of the RNA primer (Spradling, 1999; Chen, 2000; FlyBase Curators, 2004).
Furthermore, Df(3R)ED6255 (Stock# 9210) was on the border of showing a enhancement
effect, with a
ratio of 0.36. This deficiency line contained another DNA polymerase,
DNApol-α 73kD. DNApol-α 73kD plays a role in DNA binding and DNA replication
initiation (FlyBase Curators, 2004; GOA Curators, 2007). Both DNApol-α 60kD and
DNApol-α 73kD are associated with DNA-directed DNA polymerase activity, as well as
DNA-dependent DNA replication (Kuikeshoven, 1999; Cotterill, 1992). Additionally, in S.
pombe the DNA binding and pol-α binding properties, especially in the p180 subunit of
Mcm10p play an important role in DNA replication activation (Fien, 2004). This role could
transfer into Drosophila melanogaster’s Mcm10, through these subunits. The functions of
DNApol-α 60kD and DNApol-α 73kD make them likely candidates for causing the
interactions with Mcm10 seen, although other genes located in their respective deficiency
22
lines could also be creating the effects, and more specific deletion lines will be crossed to
further narrow the cause of the interactions.
Df(3L)ED4789 (Stock# 8084): Mcm10 and Reptin (Rept)
The cross between Mcm10148 and Df(3L)ED4789 produced the highest
ratio,
0.733. Upon examining the genes located in this deficiency line, Reptin stands out among the
genes identified as a candidate causing the interaction with Mcm10. Reptin is an evolutionary
conserved protein present in all eukaryotic organisms studied, and likely has a role in DNA
helicase activity, chromatin silencing and remodeling, and ATP-dependent 5’-3’ DNA
helicase activity (Grigoletto, A., 2011; Im, D. 1990; Rottbauer, W., 2002; FlyBase Curators,
2004). Drosophila Reptin participates in epigenetic processes that lead to a repressive
chromatin state through the fly TIP60 HAT complex (Qi, D. 2006). Since Mcm10 is known
to have a role in chromosome condensation and chromatin binding, it is possible an
interaction is present (Christensen, T. 2003; Christensen, T. 2002). However, due to Reptins’
role in transcription regulation and regulators does not mean it is definitively causing the
enhancement effect (Grigoletto, A. 2011). We do not yet know the function of a lot of genes
located in this deficiency line, and without further experimentation we are not able to say
with complete certainty that Rept and Mcm10 have an enhancement effect, although based
on Repts’ function it is likely.
Df(3R)ED5622 (Stock# 8959): Mcm10 and Orc2
Df(3R)ED5622 had a
ratio of 0.663. Upon examining the genes located in this
deficiency line, four stood out: Hrb87F, Orc2, CG9312, and CG9588. Little is known about
CG9312 and CG9588 other than that they are involved in the regulation of the cell cycle, and
23
cellular response to DNA damage stimulus and proteolysis, respectively. CG9312, with its
involvement in the cell cycle, could have an interaction with Mcm10. CG9588 could also
play a role in causing the effect seen, as Mcm10, in humans, is regulated by proteolysis
(Izumi, M. 2001). HRB87F is an hnRNP A1 homolog in Drosophila, likely has a role in
alternate splicing, and is transcribed maternally, decay rapidly after embryogenesis, and are
synthesized again in the late larval and pupal stages (Zu, K. 1996; Haynes, S. 1990). A
single-mutation of Hrb27c was able to be crossed with Mcm10, although the results indicated
no interaction was present. While not exactly HRB87F, it is unlikely HRB87F is interacting
with Mcm10 based upon those results. The gene most likely to be causing the enhancement
effect is Orc2, Origin recognition complex subunit 2. In Eukaryotes, origin specification and
pre-RC assembly start with the chromatin binding of ORC (Baldinger, T. 2008). Previous
work has shown interactions between Mcm10 and Orc2, and Mcm10 was shown to be
sensitive to the depletion of Orc2, with total Mcm10 protein levels being slightly reduced in
cells depleted of Orc2 (Christensen, T. 2003). Based upon the previous work, Orc2 is almost
definitely causing the enhancement effect, as the progeny with both Mcm10 and Orc2
deficiency is slightly less than either deficiencies (61 both deficiencies to 92 either
deficiency).
Df(3L)BSC816 (Stock# 27577)
Df(3L)BSC816 showed a suppression effect, with a ratio of 0.616. Within this region,
one gene could have caused the effect: hairy (h), which is involved in DNA and E-box
binding (Van Doren, M. 1994). However, Cdc6 is located on the border of this deficiency
region, and could be disrupted. Cdc6 is involved in the pre-replicative complex assembly
involved in DNA replication, and is known to associate with other Mcm complexes,
24
specifically Mcm2 and Mcm5 (Crevel, G. 2011). Due to cdc6’s role in the pre-replication
complex, it is almost certainly causing the suppression effect, despite not fully being within
the deficiency region.
Df(3L)ED201 (Stock# 8047)
Df(3L)ED201 had a final ratio of 0.00, which is likely due to some error. The total
amount of offspring was 8, with 4 CyO, 3 Sb, and 1 CyO,Sb. Upon examining the genes
located in this deficiency region, two stood out: E(Bx) and Atac3. E(bx), also known as
Nurf301, is a member of NURF, a chromatin remodeling complex that catalyzes ATPdependent nucleosome sliding (Kwon, S. 2009). It is necessary for the chromatin remodeling
required for transcription (Hamiche, A. 1999). Experiments have shown that both NURF301
and ISWI contribute to the chromatin remodeling activities of NURF (Badenhorst, P., 2002).
Based on this, an interaction between Mcm10 and Nurf301 could account for the low ratio.
Atac3 is involved in chromatin remodeling and histone acetylation, and stimulates
nucleosome sliding by the ISWI, SWI-SNF and RSC complexes (Suganuma, T.; 2008).
Finally, CG32344 is involved in helicase activity and ATP-dependent RNA helicase activity,
although little else is known of it (Lasko, P. 2000; FlyBase Curators, 2002-2003). It is worth
mentioning one other gene located in this region, due to the previous discussion of
Df(3R)BSC874: Mpk, involved in MAP kinase tyrosine/serine/threonine phosphatase
activity. Cul-5 is required for the phosphorylation of MPK-1 in the germline, and Cul-2based and Cul-5-based E3 ligases have a redundant function in meiotic cell cycle progression
through the activation of MAP kinase MPK-1 (Sasagawa, Y. 2007).
25
Df(3R)ED7665 (Stock# 8685)
When crossed with Mcm10148, Df(3R)ED7665 showed an enhancement effect, with a
ratio of 0.284. Within this deficiency line was numerous CG genes whose molecular
functions are not currently know, which could interact with Mcm10 to explain the
enhancement effect. When examining those genes whose functions are known, two stand out:
PSEA-binding protein 95kD (Pbp95) and Ubiquitin conjugating enzyme 84D (Ubc84D).
Pbp95 is involved in DNA binding, protein binding, and chromatin binding, as well as being
a member of DmPBP protein-DNA complex (Li, C. 2004; Hung, K. 2009; FlyBase Curators,
2004). Ubc84D is involved in ubiquitin-protein transferase activity, but not much else is
known (Robin, C. 1996; FlyBase Curators). It is possible Ubc84D could aid Cul-5 in the
ubiquitination of Mcm10, although Cul-5 was still active in the cross performed.
Df(3R)ED5100 (Stock# 9226)
Df(3R)ED5100 showed an enhancement effect, with a ratio of 0.299. Numerous
genes located in this deficiency line were associated with ubiquitin-protein transferase
activity, including: Skp2, Ubiquitin conjugating enzyme 6 (Ubc6), and Circadian trip (Ctrip)
(Curators, Flybase). Another gene located in this deficiency region is corto, which and is
required for proper condensation of mitotic chromosomes, having a role in the maintenance
of chromosome structure during mitosis and interphase (Kodjabachian, L. 1998). From the
genes whose functions are known, Corto is likely causing the enhancement effect shown.
Df(3R)ED5516 (Stock# 8968)
Df(3R)ED5516 showed a suppression effect, having a ratio of 0.629. This effect is likely
caused Inverted repeat-binding protein (Irbp), or a CG, whose function has yet to be
26
specifically identified. Irbp is inferred to have a function in DNA helicase activity, as well as
likely having a role in telomere maintenance, reducing the stability of terminally deficient
chromosomes (Melnikova, L. 2005; FlyBase Curators).
Df(3R)BSC568 (Stock# 25126)
The suppression effect and ratio of 0.638 is either due to error or from a CG gene
whose function has yet to be studied. Some genes in this region, such as CG6689 are inferred
to have a function in nucleic acid binding and zinc finger binding, although the specifics are
as yet unclear (FlyBase Curators, 2004).
Df(3R)BSC748 (Stock# 26846)
Df(3R)BSC748 had a final ratio of 0.652, falling into the suppression range. However, upon
examining the genes located in the deficiency region, none whose functions were known
stood out as a candidate for interaction. One gene, CG3995, has been inferred to be involved
with DNA binding, but this is not specific enough to pinpoint this gene to be causing the
suppression effect (Flybase Curators, 2004).
Df(3L)BSC12 (Stock# 6457)
Df(3L)BSC12 had a suppression effect, with a ratio of 0.653. After examining the genes
located in the region, Sneaky (Snky) could potentially cause the effect. Sneaky is involved in
sperm chromatin decondensation, fertilization, and exchange of chromosomal proteins, and
may have a role in sperm activation after entry into the egg (Fitch, K. 1998). These functions,
at a glance, would seem to cause an enhancement effect if there were no other interactions
present. At this time, there is no definite explanation for the suppression effect seen in
Df(3L)BSC12, save for error.
27
Df(3L)ED4543 (8073)
Df(3L)ED4543 had a ratio of 0.372, and was close to the edge of what ratio is
considered to have no interaction. Numerous genes stand out as possibly having an
interaction with Mcm10, especially Stonewall (Stwl), RecQ5 helicase (RecQ5), and
Trithorax-like (Trl). Stwl is a heterochromatin-associated protein able to modify chromatin,
and is likely to be required for normal compaction of chromatin, and is required to maintain
DNA integrity when replication stress is induced (Yi, X. 2009). RecQ5 is a DNA helicase in
the RecQ family, in which three are predisposed to cancer, premature aging, and
developmental abnormalities in humans, and RecQ5 has been suggested to play a role in
preventing cancer (Hu, Y. 2007). RecQ5 maintains genome stability through participating in
many DNA metabolic processes, including DNA repair and DNA resolution, which overlaps
with functions Mcm10 likely performs (Sakurai, H. 2013). Of the three genes isolated,
Trithorax-like is the least likely to have an interaction with Mcm10. Trl encodes the GAGA
factors, which is a multifunctional protein involved in gene activation, Polycomb-dependent
repression, chromatin remodeling, and is a component of chromatin domain boundaries
(Chopra, V. 2008).
Df(3R)ED10555 (23714)
Df (3R)ED10555 was on the border of an enhancement effect, with a ratio of 0.375. Based on
the functions stated by Flybase, three genes stand out: BigH1, Eff, and His4r. BigH1 is
involved in regulating nucleosome density and assembly, and is known to be located in
chromatin. Eff, meanwhile, is involved in ubiquitin conjugating enzyme activity, ubiquitin
protein ligase activity, and ubiquitin protein ligase binding, as well as chromosome
organization. Finally, His4r is predicted to have a role in centrosome duplication, chromatin
28
assembly or disassembly, as well as nucleosome assembly. Of the three, His4r is least likely
to be causing the low ratio shown through the cross, although it is as yet undetermined.
Df(3R)ED10845 (Stock# 9487)
Df(3R)ED10845 was also on the edge of an enhancer effect, with a ratio of 0.378. Two genes
located within this region have roles ubiquitin processes: Usp8 and Slmb. Slmb is inferred to
play a role in regulation of chromosome condensation and ubiquitin-protein transferase
activity, functioning as a member of the SCF ubiquitin ligase complex. The SCF complex has
a catalytic core consisting of a cullin from the Cul1 subfamily and a RING domain protein
(Willems, AR. 2004). Usp8, meanwhile, is involved in ubiquitin-specific protease activity,
protein deubiquitination, and is found in the cytoplasm (Xia, R. 2012). Based upon this, Slmb
is the gene most likely to be the cause of the near-enhancement effect observed.
Df(3R)ED5330 (Stock# 9077)
Finally, Df(3R)ED5330 was also on the edge of an enhancement effect, with a ratio of 0.387.
Numerous genes could be contributed to this, including: Neur, pyd, Kdm2, and E(var)3-9.
Based upon the functions stated by Flybase, Neur is involved in ubiquitin protein ligase
activity, protein polyubiquitination, zinc ion binding, and DNA binding. Pyd is also involved
in ubiquitin protein ligase binding. Both Neur and Pyd are involved in the Notch signaling
pathway, and it is possible the loss of these two genes could cause the low ratio. Both of the
offspring genotypes containing the deficiency line were lower than the offspring containing
only the Mcm10148 mutation, as shown in Figure 5 below.
29
Figure 5. Graphical representation of the progeny from Df(3R)ED5330 x Mcm10148. The
Wt offspring contain both the Df(3R)5330 and Mcm10 mutations. The CyO offspring contain only the
Df(3R)ED5330 mutant, and Sb contains only the Mcm10 148 mutation.
Kdm2 is involved in histone demethylase activity, ubiquitin-protein transferase activity, zincion binding, and is inferred to be a member of the SCF ubiquitin ligase complex, encountered
earlier (FlyBase, 2008). Finally, E(var)3-9 plays a role in chromatin maintenance and
structure determination, and encodes a zinc finger protein (Weiler, 2007).
30
Conclusion
From previous studies, we know Mcm10 may have separate roles in DNA replication,
endoreplication, the cell cycle, DNA damage repair, origin firing, and stem cell maintenance.
Based upon the screen performed, Mcm10 may interact with many different proteins
involved in similar functions. Many genes identified correlate with previous studies
performed on Mcm10’s function. DNA polymerase-α60kd and DNA polymerase-α73kd
stand out as genes likely causing the interactions shown in their deficiency lines based upon
agreement with the literature. Other genes, such as Reptin or E(bx), a member of the NURF
chromatin remodeling complex, are not as certain to be causing the interactions, although due
to their similar functions to Mcm10 they are likely involved with Mcm10 in some way. Of
particular interest is Cul-5, a ubiquitinating protein known to associate with E3-ligases,
which was shown through single-mutant crosses to have a suppression effect on the Mcm10
mutant. The identification of this interaction increased the likelihood of other genes involved
in the ubiquitination process to be interacting with Mcm10. One example of this is Neur,
from Df(3R)ED5330, which is known to be involved in protein polyubiquitination, DNA
binding, and in ubiquitin protein ligase activity. Without the discovery of the interaction
between Cul-5 and Mcm10, Neur would not be as likely of a candidate to be the cause of the
likely interaction present in Df(3R)ED5330. However, no single gene can be determined to
be interacting with Mcm10 without more specific studies. Further analysis of shorter
deficiency lines, as well as with single gene mutations, is necessary to pinpoint the specific
genes causing the suppression and enhancement effects demonstrated in the crosses.
31
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