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University of Tennessee, Knoxville
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Exchange
Masters Theses
Graduate School
8-2007
Studies on the Mechanisms of Homolog Pairing
and Sister Chromatid Cohesion During
Drosophila Male Meiosis
Jian Ma
University of Tennessee - Knoxville
Recommended Citation
Ma, Jian, "Studies on the Mechanisms of Homolog Pairing and Sister Chromatid Cohesion During Drosophila Male Meiosis. "
Master's Thesis, University of Tennessee, 2007.
http://trace.tennessee.edu/utk_gradthes/167
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To the Graduate Council:
I am submitting herewith a thesis written by Jian Ma entitled "Studies on the Mechanisms of Homolog
Pairing and Sister Chromatid Cohesion During Drosophila Male Meiosis." I have examined the final
electronic copy of this thesis for form and content and recommend that it be accepted in partial
fulfillment of the requirements for the degree of Master of Science, with a major in Biochemistry and
Cellular and Molecular Biology.
Bruce D. McKee, Major Professor
We have read this thesis and recommend its acceptance:
Hong Guo, Mariano Labrador
Accepted for the Council:
Dixie L. Thompson
Vice Provost and Dean of the Graduate School
(Original signatures are on file with official student records.)
To the Graduate Council:
I am submitting herewith a thesis by Jian Ma entitled: “Studies on the mechanisms of
homolog pairing and sister chromatid cohesion during Drosophila male meiosis”. I
have examined the final electronic copy of this thesis for form and content and
recommend that it can be accepted in partial fulfillment of the requirement for the
degree of Master of Science, with a major in Biochemistry, Cellular and Molecular
Biology.
McKee, Bruce D., Ph.D., Major Professor
We have read this thesis
and recommend its acceptance:
Guo, Hong, Ph.D.
Labrador, Mariano, Ph.D.
Accepted for the Council:
Carolyn R. Hodges, Vice Provost and
Dean of the Graduate School
(Original signatures are on file with official student records.)
Studies on the mechanisms of homolog
pairing and sister chromatid cohesion
during Drosophila male meiosis.
A Thesis
Presented for the
Master of Science Degree
The University of Tennessee, Knoxville
Jian Ma
August 2007
ACKNOWLEDGEMENT
This thesis could not be finished without the help and support of many people
who are gratefully acknowledged here.
I am honored to express my deep and sincere gratitude to my major supervisor,
Professor McKee, Bruce, Ph. D., Director, Department of Biochemistry, Cellular and
Molecular Biology, the University of Tennessee. He has offered me valuable ideas,
suggestions and criticisms with his profound knowledge in linguistics and rich
research experience. His understanding, encouraging and personal guidance have
provided a good basis for the present thesis.
I am extremely grateful to Assistant Professor Guo, Hong and Assistant
Professor Labrador, Mariano, Department of Biochemistry, Cellular and Molecular
Biology, the University of Tennessee. Their ideals and concepts have had a
remarkable influence on my thesis.
I also wish to thank my lab members: Thomas, Sharon; Matulis, Shannon; Yan,
Rihui; Tsai, Jui-He, and Vandiver, Jennifer. Their kind support has been of great value
in this study.
I owe my loving thanks to my wife Shengli Ding and my son Brian E. Ma.
Without their encouragement and understanding it would have been impossible for me
to finish this work.
ii
ABSTRACT
Meiosis is a complex process involving one round of DNA replication
followed by two rounds of cell divisions. The proper segregation of homologs at
meiosis I and sister chromatids during meiosis II is essential for the survival of the
offspring. Aberrant chromosome segregation at any stage of meiosis can lead to
aneuploidy. Meiotic chromosome segregation without crossing over or chiasmata is a
widespread but poorly understand chromosome segregation pathway. In male
Drosophila meiosis the absence of recombination in chromosomes makes it easier to
identify mutations which influence homologous chromosome pairing and segregation.
Modifier of Mdg4 in Meiosis (MNM), a protein encoded by modifier of mdg4,
is required for integrity of chromosome territories and stability of achiasmatic
bivalents and for normal homolog segregation in male Drosophila meiosis I. MNM
localizes to clusters of nucleolar and autosomal foci during meiotic prophase I (PI)
and to a novel, compact structure associated with the X-Y bivalent during
prometaphase I (PMI) and metaphase I (MI). Stromalin in Meiosis (SNM), a member
of the SCC3/STAG cohesion family, is required for homolog pairing in male
Drosophila but not for meiotic sister chromatid cohesin. SNM protein co-localizes
with MNM to the nucleolus throughout PI and to a prominent focus on the X-Y
bivalent during PMI and MI. Mutations of snm and mnm exhibit similar homolog
pairing failure during meiosis I. Consequently we used the Yeast Two-Hybrid System
to determine whether SNM and MNM can interact with each other. We concluded that
MNM can interact with itself and SNM. We also found that SNM interacts with the
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BTB domain of MNM and that the FLYWCH domain in the C-terminus of the MNM
protein may play a role in the interaction between MNM and SNM.
Sister chromatid cohesion (SCC) is required for proper chromosome
segregation during mitosis and meiosis. The protein complex cohesin is a major
component of SCC and links sister chromatids together from the time of their
replication until their segregation. sisters unbound (sun) is a novel gene required in
male and female Drosophila for meiotic SCC. Mutations in sun cause premature sister
chromatid segregation (PSCS) and nondisjunction (NDJ) of both homologous and
sister chromatids, and also disrupt normal recombination and synapsis in female
meiosis. The four chromatids in each bivalent exhibit random segregation at meiosis I.
We found that centromeric cohesion is lost in the absence of SUN during
mid-prophase (S4). Surprisingly, cytological analysis shows chromosome behavior
appears relatively normal during meiosis I.
Double mutations sun snm and sun mnm impair the integrity of chromosome
territories. In addition we found that SNM, but not MNM, is required for centromere
pairing in mid-prophase (S3) and simultaneous loss of SNM and SUN proteins causes
PSCS at mid-prophase I (S3), which is earlier than in single mutants in snm or sun.
These findings indicated that these two proteins play complementary roles in meiotic
cohesion.
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TABLE OF CONTENTS
Chapter I – General Introduction
1
I-1 Overview of Meiosis………………………………………………….……..……2
I-2 Meiotic Recombination and Chiasma………………………………………….…4
I-3 Cohesion and Cohesin………………………………………..………..……….…5
I-4 Meiotic Cohesion……………………………………………………….………...12
I-5 Meiosis in Drosophila……………………………………….…………………...16
I-6 Meiotic Cohesion in Drosophila…………………………..…………………….. 21
Chapter II – Evidence for a homolog conjunction complex in Drosophila male
meiosis
24
II-1 Introduction……………………………………………………….…………..…25
II-2 Materials and Methods……………………………………………………..……29
II-3 Results and Discussion…………………………………………….…………….36
Chapter III – sun is required for sister chromatid cohesion and sister centromere
mono-orientation in male Drosophila meiosis
45
III-1 Introduction…………………………………………………………………….46
III-2 Material and Methods……………..…………………………….…………...…48
III-3 Results…………………………………………………………..………………50
III-4 Discussion…………………………………………………….….……………..59
Chapter IV – Roles of interaction of sun, snm and mnm in meiotic cohesion,
centromere pairing and chromosome territory integrity
62
IV-1 Introduction………………………………………………………….…………63
IV-2 Materials and Methods…………………………………..………….….………64
IV-3 Results……………………………………………………………….…………65
IV-4 Discussion…………………………………………………………….……..…73
Chapter V – References
76
Vita
102
v
List of Figures
Chapter I – General Introduction
Figure I-1: Model of Meiosis
Figure I-2: Model of chiasmata
Figure I-3: Model for ATP hydrolysis-dependent binding of cohesin to DNA
Figure I-4: Model for release of sister chromatid cohesion in meiosis
Figure I-5: Model for meiotic pairing in male Drosophila
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6
9
14
20
Chapter II – Evidence for a homolog conjunction complex in Drosophila male
meiosis
Figure II-1: Structures of the pGAD-C and pGBD-C vectors
Figure II-2: Structure of BTB and FLYWCH domain of MNM protein
Figure II-3: MNM interact with itself and with SNM
Figure II-4: β-gal activity for MNM and SNM interaction
Figure II-5: BTB domain interact with SNM
Figure II-6: Comparison of β-gal activities
30
32
37
40
42
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Chapter III – sun, a novel gene, is required for sister chromatid cohesion and
sister centromere mono-orientation in male Drosophila meiosis
Figure III-1: Molecular characterization of sun
Figure III-2: NDJ test of homologs vs sister chromatids for sun mutants
Figure III-3: Chromosome morphology during meiosis I in sun mutants
Figure III-4: Sister chromatids segregate in meiosis II in sun mutants
Figure III-5: sun mutants and wild-type in early-prophase (S1/2)
Figure III-6: sun mutants and wild-type in mid-prophase (S3)
Figure III-7: sun mutants and wild-type in mid-prophase (S4)
Figure III-8: sun mutants and wild-type in mid-prophase (S5)
47
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57
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Chapter IV – Roles of interaction of sun, snm and mnm in meiotic cohesion,
centromere pairing and chromosome territory integrity
Figure IV-1: Chromosome morphology in meiosis I in sun snm mutants
Table IV-2: Sperms produced by sun mutants & sun snm mutants
Figure IV-3: Sister centromere cohesion is lost in sun snm double mutants
Figure IV-4: Comparison of > 8 CID spots percentage in prophase I
Figure IV-5: Chromosome territories in PI in sun snm & sun mnm mutants
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Chapter I - General Introduction
1
I-1 Overview of Meiosis
Meiosis, conserved in eukaryotes, is a special cell division which allows for
the exchange of genetic material between parental chromosomes to maintain the
genetic diversity of offspring. Meiosis comprises a round of DNA replication
followed by two successive nuclear divisions, meiosis I and meiosis II (Kleckner,
1996). Meiosis I is a reductional segregation in which homologous chromosomes pair
and segregate to opposite poles but sister chromatids stay together. In meiosis II, an
equational division which resembles a mitotic division, sister chromatids separate and
move to opposite poles. After meiosis a diploid germ cell produces four haploid cells
containing half number of chromosomes (Fig.I-1). Meiosis is important for the correct
propagation of species in all sexually reproducing organisms and for the diversities of
genome and phenotype. Any errors that affect meiosis, such as mutations in homolog
pairing or sister chromatid cohesion pathways, can lead to homologous chromosome
or sister chromotid nondisjunction (NDJ) and produce aneuploidy, which is the
leading cause of genetic illnesses such as Down's syndrome and human spontaneous
abortion (Hassold et al., 2001). Considering the significance, it is necessary to
uncover the mechanisms of homologous chromosome pairing and disjunction and
sister chromatid disjunction.
Meiosis I and II are both divided into four phases: prophase, metaphase,
anaphase, and telophase. Prophase I (PI) is the first stage of meiosis I, during which
several important changes in chromatin architecture should take place. First of all,
each individual chromosome condenses and lines up with its homologous
2
---Brown, 2002
Figure I-1: Model of Meiosis. One member of the pair is red, the other is blue. Image
from Brown, 2002.
3
chromosome, generating bivalents. Second, the synaptonemal complex (SC), a protein
structure consisting of two parallel lateral regions and a central element, forms
between two homologous chromosomes to mediate chromosome pairing and
recombination (crossing-over) (Cohen et al., 2001). The pachytene substage of PI
ends when the SC disappears, and nuclear envelope breakdown marks the start of
prometaphase I (PMI). During metaphase I (MI) the condensed homologs are
arranged on the metaphase plate. Three important events need to occur prior to MI to
ensure that homologs segregate faithfully: a physical linkage between homologous
chromosomes has to be established to resist the force from the opposite poles; sister
chromatids must be held together beyond meiosis I by a protein complex termed
cohesin; sister kinetochores have to attach to microtubules from the same pole to
result in a mono-oriented movement (Brian et al,. 2001). Any error within one of
these three events will cause homolog NDJ or PSCS in meiosis I. Segregation of
homologs to opposite poles initiates at anaphase I (AI) with the release of homolog
connection, and formation of two daughter cells at telophase I (TI) concludes meiosis
I. Meiosis II has the same four phases as meiosis I and produces four daughter cells
with half the number of chromosomes after sister chromatid segregation.
I-2 Meiotic Recombination and Chiasma
As mentioned above, in the majority of eukaryotic organisms, recombination,
or the formation of crossovers is important for proper segregation of homologous
chromosomes at MI, as well as providing genetic diversity. Meiotic recombination is a
4
virtually universal feature in most organisms and is initiated via programmed double
strands break (DSBs) catalyzed by the topoisomerase-like protein Spo11. Several
other proteins including the RAD50/ MRE11/ XRS2 complex also have been proved
to involve in the formation of DSBs and in resection of the 5’ strand termini to give
molecules with ~ 300 nt 3’ single-stranded tails (Keeney et al., 1997; Bergerat et, al.,
1997; Keeney et al., 2001; Neale et al., 2005). The majority of these DSBs are
repaired via homologous recombination with the homologous sequence on a
homologous chromatids, rather than with the sister chromatids. A fraction of these
recombination events proceeds by two long-lived intermediates: single-end invasions
(SEIs) and double Holliday junction (dHJs) to result in crossovers and chiasmata,
physical connections between homologous chromosomes (Fig. I-2) (Hunter et al.,
2001; Allers et al., 2001). The chiasma is an important apparatus for homolog
segregation in meiosis I. It holds homologs together after SC is removed and provides
the force to resist the pulling power from the opposite poles to prevent the
homologous chromosomes from separating prior to AI (Zickler et al., 1999)
I-3 Cohesion and Cohesin
Sister chromatid cohesion was first named in 1994 (Miyazaki et al., 1994) to
refer a physical linkage between two duplicated sister chromatids. It is established
from the time of DNA replication in S-phase and holds sister chromatids together
through chromosome arms and the centromeres. Sister chromatid cohesion resists the
power of microtubules from the opposite poles while aligned at the metaphase plate
5
Figure I-2: Model of chiasmata. Image from Griffiths, 1999
Each line represents a chromatid of a pair of synapsed chromosomes.
6
and is released to allow sister chromatid segregation during anaphase of mitosis or
meiosis II. In addition, sister chromatid cohesion has been proved to hold homologs
together in meiosis I by stabilizing chiasmata (Lee et al., 2001), and is involved in
repair of DNA double-strand breaks during G2 phase (Sjogren 2001). The molecular
basis for sister chromatid cohesion is a chromosomal protein complex, called cohesin.
Cohesin is a multi-subunit complex containing at least four conserved proteins: SMC1,
SMC3, SCC1 (Also known as MCD1 or RAD21) and SCC3 (also known as SA in
vertebrate cells) from yeast to human. SMC1 and SMC3 are members of Structural
Maintenance of Chromosomes (SMC) proteins, a superfamily that has multiple
functions in regulating the structural and functional organization of chromosomes
from bacteria to human, such as chromosome and sister chromatid segregation (during
mitosis and meiosis), chromosome condensation, chromosome-wide gene regulation
and DNA recombinational repair (Losada et al., 2005). All SMC proteins have a
conserved characteristic architecture in which they form long coiled-coil arm between
a ‘hinge’ domain at one end and an ABC-type ATPase ‘head’ domain at the other by
folding back on themselves through an antiparallel coiled-coil interaction. The ‘head
domain’ can close and open respectively by the ATP binding and hydrolysis (Hirano
et al., 2001; Haering et al., 2002; Arumugam et al., 2003; Hirano, 2006). Cohesin
exhibits a tripartite ring structure, within which SMC1 and SMC3 form a V-shaped
molecule structure through the association with each other at their hinge domains, and
SCC1, which belongs to the kleisin superfamily representing the most conserved
SMC-interacting proteins, closes this ring structure by binding to the head domains of
7
SMC1 and SMC3 through its C- and N-terminal domains respectively. The fourth
subunit SCC3 binds to the ring through its interaction with SCC1 (Gruber et al., 2003;
Schleiffer et al., 2003). Ring-like cohesin is thought to hold sister chromatids together
by surrounding and entrapping them, but direct proof is absent. There are several
models to explain how cohesin embraces the sister chromatids. One model is that the
entry of DNA into cohesin’s ring is based on the ABC-type ATPase ‘head’ domain of
SMC proteins (Fig. I-3). Hydrolysis of ATP opens the ring by destroying the
interaction of the head domains and allows the DNA to slide inside, and binding of a
new ATP molecule then re-closes the ring to embrace the DNA (Arumugam et al.,
2003; Weitzer et al., 2003). Recently Gruber et al. proposed another model in 2006
within which the SMC hinge domain is responsible for the entry of DNA into
cohesin’s ring. The transient dissociate of the hinge-hinge interface of the
SMC1-SMC3 allows the DNA to enter the cohesin ring (Gruber et al., 2006).
In yeast cohesion is established during S phase. Cohesin is loaded onto
chromosomes with help of the loading complex SCC2 and SCC4 (Ciosk et al., 2000),
but the establishment of cohesion in S phase requires several other proteins, such as
ECO1/CTF7 (Skibbens et al., 1999; Toth et al., 1999), TFR4 and TRF5 (Wang et al.,
2000). Milutinovich proved that the hinge and loop1 regions of SMC1 also play an
important role in the binding of cohesin to the specific chromatin sites including
cohesion-associated regions (CRAs) and pericentric regions (Milutinovich et al.,
2007). In addition, condensin has been found to be required to maintain cohesion at
several chromosomal arm sites, but not required at centromere (Lam et al., 2006).
8
---Uhlmann, 2004
Figure I-3. A model for ATP hydrolysis-dependent binding of cohesin to DNA. The
SCC1 binds to the SMC heads to form a ring structure. ATP hydrolysis leads to
separation of the SMC heads and let DNA enter the ring structure. When all
chromosomes are aligned at metaphase plate, Separase is activated to cleave SCC1
and open the ring; leading to removal of sister chromatid cohesion.
9
Cohesin along the chromosome arms is at lower density (Lengronne et al., 2004;
Glynn et al., 2004), but enriched around centromeres. The heterochromatin protein
HP1/Swi6 at pericentromeric regions is thought to promote the centromeric
enrichment of cohesion, presumably through direct interaction with the cohesin
subunit Scc3/SA (Pidoux et al., 2004; Ishiguro et al., 2007).
The removal of cohesin is a vital step for the faithful segregation of
chromosomes in both mitosis and meiosis. This removal is triggered by proteolytic
cleavage of SCC1/RAD21 by Separase at the onset of anaphase in mitosis. Separase is
a cysteine protease which is inhibited, for most of the cell cycle, by binding its
inhibitor Securin. Once all chromosomes have been bioriented during metaphase,
Securin is marked for degradation by a ubiquitin ligase called the Anaphase
Promoting Complex or Cyclosome (APC/C). Securin degradation releases Separase
allowing it to destroy cohesin and trigger the onset of anaphase (Cohen-Fix et al.,
1996; Funabiki et al., 1996b; Ciosk et al., 1998; Uhlmann et al., 1999; Zou et al.,
1999). In vertebrate cells, Separase activity is inhibited by not only Securin but also
its phosphorylation at the hands of Cdk1 kinase. In these cells, the destruction of
Securin and proteolysis of the CDK1 subunit cyclin B simultaneously, both mediated
by APC/C, will activate Separase (Stemmann et al., 2001).
In contrast to the simultaneous release of cohesion from the chromosome arms
and centromeres in budding yeast, in vertebrate cells dissociation of cohesin from
chromosomes is carried out in two steps (Waizenegger et al., 2000). The first step is to
release cohesin from chromosome arms before metaphase by hyperphosphorylation of
10
SCC3/SA subunits mediated by the Polo-like kinase 1 (PLk1) and Aurora B without
SCC1 cleavage. This process is called the ‘prophase pathway’ (Gimenez-Abian et al.,
2004; Sumara et al., 2002). Hauf found in 2005 that SA2 is the critical target of Plk1
in this pathway (Hauf et al., 2005). However, centromeric cohesin still persists and is
removed at the metaphase-anaphase transition by Separase, which is the second step.
The protection of centromeric cohesin from the prophase pathway is
accomplished by the centromeric protein Shugoshin (Sgo), an ortholog of the
MEI-S332 protein from Drosophila melanogaster (Watanabe, 2005; Lee et al., 2005).
Sgo was found in 2004 by genetic screening in budding and fission yeast as a
protector of centromere cohesion in mitosis and meiosis. So far, budding yeast, worm
and Drosophila possess only a single orthologue, Sgo1 and MEI-S332, respectively,
that is expressed in mitotic as well as meiotic cells. Fission yeast and humans have
two Sgo proteins. Researchers found that human Sgo1, hSgo1, localizes to centromere
in mitosis in the presence of Bub1, a spindle checkpoint protein, to prevent the
premature centromeric cohesin dissociation, and disappears during anaphase. Three
recent papers bring new light on the molecular mechanism of this protection:
phosphatase 2A (PP2A), a major partner of Sgo in human and yeast cells, is involved
in centromeric cohesin protection. PP2A colocalizes with Sgo at the centromere by
binding its PP2A-B’ subunit to Sgo’s N-terminal region from prophase to anaphase in
the presence of Bub1 and counteracts the phosphorylation of cohesion subunits by
PLK1. In addition, Tang et al. found in 2006 that although PP2A is required for the
centromeric localization of hSgo1 and for counteracting the phosphorylation of hSgo1
11
by Polo kinase, which would otherwise result in removal of hSgo1 from chromosome,
hSgo1 may also have a PP2A-independent function on the protection of centromeric
cohesin because the precocious sister chromatid separation phenotype of
PP2A-deficient human cell, but not that of hSgo1-deficient cells, can be rescued by
the depletion of Polo kinases. hSgo2 is another human Sgo protein. Kitajima
concluded that hSgo2 has a role in PP2A recruitment to human mitotic chromosomes
(Kitajima et al., 2006; Riedel et al., 2006; Tang et al., 2006). Another interesting study
showed that neither PLK1 depletion nor Aurora B inhibition suppresses PSCS in the
absence of hSgo1, indicating that additional proteins could contribute to the prophase
dissociation pathway (McGuinness et al., 2005). In addition, S. cerevisiae Sgo1 plays
a role in the bi-polar attachment of kinetochores by activating the spindle checkpoint,
which indicates a molecular link between sister chromatid cohesion and
tension-sensing at the kinetochore-microtubule interface (Indjeian et al., 2005).
I-4 Meiotic Cohesion
Sister chromatid cohesion is also required for both meiotic divisions, but differs
in several ways from its role in mitosis. In meiosis I, cohesion is involved in at least
three unique functions. First, cohesion along chromosome arms promotes
recombination between homologous chromosome and the formation of synaptonemal
complexes (SCs) (Revenkova et al., 2004). Second, cohesion close to crossovers
stabilizes chiasmata and is required for the disjunction of homologues in meiosis I
(Klein et al., 1999; Petronczki et al., 2003). Third, cohesion functions in
12
mono-orientation of sister centromeres. In meiosis II, cohesion has similar roles to
mitosis, including providing tension to promote sister chromatids alignment and
holding sister centromeres together until anaphase II (AII).
The most important change in the cohesin complex between meiosis and
mitosis is that Scc1/Rad21 in mitosis is replaced largely by a meiotic-specific paralog
Rec8 (Watanabe et al., 1999; Stoop-Myer et al., 1999; Kitajima et al., 2003). This
Rec8 replacement may promote DNA recombination, synaptonemal complex
formation, monopolar attachment and persistent centromeric cohesion until to AII.
However, in budding yeast, most of these meiosis-specific properties can be
substituted by Scc1/Rad21 except for the maintenance of sister centromere cohesion
until AII (Toth et al., 2000; Yokobayashi et al., 2003; Xu et al., 2005). In addition,
other meiosis-specific cohesin subunits have been found in various organisms. For
example, in S. pombe, PSC3, which is the SCC3 subunit, is replaced in some meiotic
cohesin complexes by a meiosis-specific counterpart, Rec11. Budding yeast Spo13, a
novel protein, does not localize to the centromere, but is crucial for retention of
centromeric cohesin in meiosis and when ectopically expressed in mitosis it can
inhibit release of cohesin (Lee et al., 2002; Shonn et al., 2002).
In budding yeast, sister chromatid cohesion is disrupted in two-step process
in meiosis by the cleavage of Rec8 on chromosome arms at AI and at centromeres at
AII by Separase, the same enzyme that cleaves the mitotic cohesin Scc1/Rad21 (Fig.
I-4) (Buonomo et al., 2000). First, Rec8 along the chromosome arm is cut, resulting in
disassociation of cohesin from arms. In addition, condensin and Cdc5, a Polo-like
13
--- JESSBERGER, 2005
Figure I-4. A model shows that SCC in meiosis is released in two steps. At meiosis I,
the homologs move toward opposite poles, while the sister chromatids of each
homolog remain connected because of centromeric cohesin. In meiosis II, centromeric
cohesin is removed, resulting in sister chromatid segregation.
14
kinase, also can facilitate the removal of cohesin from chromosome arms before AI
(Yu et al., 2005). However sister chromatid cohesion is preserved at centromeres
throughout AI until MII due to the protection of centromeric Rec8 by the proteins
Shugoshin (Sgo1) and PP2A phosphatase which shield centromeric cohesin from Polo
kinase-dependent removal by dephosphorylating Rec8, as in mitotic animal cells
(Kitajima et al., 2006; Riedel et al., 2006). Another protein involved in the protection
of centromeric cohesion is Bub1 which is involved in protection of centromeric
cohesin by recruiting Sgo to centric localization (Tang et al., 2004; Hamant et al.,
2005; Kiburz et al., 2005; Kitajima et al., 2005). Mutations in each of the above genes
cause PSCS during meiosis I. Second, centromeric Rec8 is cleaved by Separase at the
onset of AII, resulting in sister chromatids segregating into each daughter cell.
During meiosis I sister chromatids of a homolog develop potentially two
independent kinetochores. However each sister kinetochore must attach to
microtubules emanating from same spindle pole (monopolar attachment), instead of
opposite poles, so that sister chromatids can move to the same pole at meiosis I.
Although the mechanism of sister kinetochore mono-orientation is poorly understood,
some research showed that centromeric cohesin complexes contribute to this
monopolar attachment by holding sister kinetochores together. One line of evidence is
that in fission yeast, mutations in the meiosis-specific cohesin subunit Rec8 result in
bipolar attachment of sister kinetochores in meiosis I (Watanabe 1999; Yokobayashi et
al., 2003; Yamamoto et al., 2003; Hauf et al., 2004). In addition, Monopolin in S.
cerevisiae and Moa1 in S. pombe have been proved to be required for
15
mono-orientation of sister chromatids in meiosis I and Moa1 can interact with Rec8
(Toth et al. 2000; Yokobayashi et al. 2004). However, in both organisms Rec8 cohesin
complex also play a role in the monopolar attachment of sister kinetocores, and loss
of Rec8 function causes a random chromatid orientation (S. cerevisiae), or an
equational orientation (S. pombe) at meiosis I (Klein et al., 1999; Watanabe et al.,
1999).
I-5 Meiosis in Drosophila
Meiosis in Drosophila as a model has been investigated for several decades
because of its several advantages. The first is that scientists can visualize the meiotic
divisions in both male and female meiosis. The second is that there are a valuable
collection mutations affecting meiosis. The third is Drosophila has a short life cycle
and only four paired bivalents. The fourth is that the mechanisms of male and female
Drosophila meiosis systems are different. Female Drosophila has both chiasmate and
achiasmate meiotic mechanisms. The three large chromosomes have chiasmata to
stabilize the interhomolog connections during meiosis and mutations affecting
recombination can cause aberrant homolog segregation. For instance, mutations in
homologous recombination genes including rad51/spindle-A (spnA), spindle-B (spnB),
spindle-D (spnD) and okra have been found to affect meiotic recombination in
females, and cause a reduction in recombination and increase of NDJ (Ghabrial et al.,
1998; Morris et al., 1999, Yoo et al., 2004). However, these three large chromosomes
can also segregate correctly using an achiasmate pathway termed distributive
16
segregation if exchange is suppressed by heterozygosity for multiple inversions. The
tiny fourth chromosomes which never recombine always segregate via the distributive
pathway (Orr-Weaver 1995).
Male Drosophila has a unique meiotic system in which recombination does
not occur and SC and chiasmata are not detectable. Nevertheless, these non-exchange
homologs can pair and segregate faithfully during meiosis I. Mutations in genes
affecting the exchange segregation pathway in females, such as the Spo11 homolog
mei-W68 or spnA, spnB, spnD and okr, do not affect male meiosis. Also, mutations in
genes required for the non-exchange distributive pathway such as nod (no distributive
disjunction) and ncd (nonclaret disjunctional) have no effect on male meiosis
(Knowles et al., 1991; Orr-Weaver 1995). Thus genes neither affecting recombination
nor distributive segregation in female can explain the mechanism of male meiosis. In
addition, mutations in the SC gene c(3)G (crossover suppressor on 3 of Gowen)
abolish both SC formation and meiotic recombination in female Drosophila but does
not alter the male homologs segregation pattern (Walker et al., 2000; McKim et al.,
2002; McKee, 2004). These data strongly support that male meiosis in Drosophila
lacks crossovers and involves a different meiotic segregation system. However, it is
unclear how homologs pair in male Drosophila and what kind of apparatus provides
the power to hold homologs together and balance the forces from opposite poles.
Cooper demonstrated in 1964 that the X and Y chromosome pair at specific sites,
thread-like structures of heterochromatin known as collochores near the nucleolus
organizers (NORs), where the repeated genes for the 18s and 28s are located (Cooper
17
et al., 1964). Mckee and coworkers proved a 240 bp repeated sequence within the
intergenic spacer of the ribosomal DNA (rDNA) can mediate disjunction of the X and
Y chromosomes and the efficiency of segregation is dependent on the copy number of
the intergenic spacer. But the autosomes do not contain any rDNA, which means they
likely have multiple pairing sites accounting for their segregation (McKee et al., 1992;
McKee et al., 1993; McKee, 2004). Currently, only a few genes have been identified
to affect homolog segregation and cause meiosis I NDJ in male Drosophila including
the male-specific genes, teflon (tef) and mei-s8, both of which localize to the 2nd
chromosome. The teflon gene is required for disjunction of all autosomes, but not for
sex chromosomes (Tomkiel et al., 2001). Thomas et al., 2005 identified two novel
genes on the 3rd chromosome: stromalin in meiosis (snm) and modifier of mdg4 in
meiosis (mnm). These two genes are required for segregation of all homolog pairs in
male Drosophila meiosis I, but are dispensable for female meiosis and for sister
chromatid segregation. Mutations in snm and mnm disrupt homolog conjunction and
result in high frequencies of homolog NDJ in male Drosophila, but premeiotic and
meiotic homolog pairing is normal. Using antibodies against SNM and MNM, MNM
and SNM proteins were found in the nucleolus during PI and localize co-dependently
to the X-Y bivalent pairing site during late PI through MI and disappear at AI. SNM is
independent of MNM but MNM depends upon SNM with respect to their nucleolar
localization. Using an MNM-GFP construct that fully rescues mnm mutations, MNM
was also found to localize to multiple autosomal foci throughout meiosis I and this
autosomal binding was found to depend on the teflon gene (Tomkiel et al., 2001,
18
Thomas et al., 2005). Recently, a Venus-tagged SNM protein that fully rescues snm
mutations has been found to localize to autosomal foci as well as to the nucleolus and
X-Y bivalent (S Thomas. personal communication).
Another distinguishing character of meiosis in male Drosophila is meiotic
prophase I (PI), a growth phase during which the primary spermatocytes undergo an
approximate 25-fold increase in volume, lasts approximately 90 hours and is divided
in substages S1-S6 depending on size of the nucleus and chromatin architecture
(Cenci et al., 1994).
In stages S1 and S2 (early-G2), the nucleus lies in an eccentric position within
the cytoplasm and the chromatin positions at the center of the nucleus as a compact
mass. Homologous chromosomes are tightly paired at this stage via pairing sites
present in the euchromatin (Vazquez et al., 2002). When spermatocytes enter the late
S2b or early S3, the chromatin mass subdivides into three main territories, presumably
corresponding to the three major bivalents: XY, 2nd, and 3rd (Vazquez et al., 2002).
Interestingly, euchromatic pairing is dissolved at the time (S2b/S3) that are
established. These homologous chromatin masses remain at the inner nuclear
envelope from mid-prophase I (S3-S4) until the onset of PMI. Then the chromosomes
condense very rapidly which marks the onset of MI (Fig. I-5). Following two cell
divisions, and a series of dramatic morphological changes, the resulting spermatids
contains the half of the original eight chromosomes (Fig. I-5) (Cenci et al., 1994).
19
---Vazquez et al., 2002
Figure I-5. A model for meiotic pairing in male Drosophila
(A) A summary of spermatocyte development. Shading indicates chromatin. The
formation of chromosome territories marks the beginning of stage S3 in mid-G2.
(B) A model for meiotic chromosome pairing in male Drosophila. Forming the
distinct territories represents the beginning of stage S3.
20
I-6 Meiotic Cohesion in Drosophila
Drosophila has been a good model for genetic research for many years, but the
mechanism and functions of meiotic cohesion are still an enigma because of no
mutations in cohesin genes. The Drosophila genome contains single SMC1 and
SMC3 genes and two members each of the SCC1 (RAD21 and C(2)M) and SCC3/SA
(SA and SNM) families. C(2)M), although it exhibits weak similarity to SCC1 and
REC8, is a synaptonemal complex component and functions only in synapsis and
recombination during PI in females, not in centromeric cohesion in either sex
(Manheim et al., 2003; Heidmann et al., 2004; Anderson et al., 2005). SNM identified
by Thomas et al. in 2005 as a new meiosis-specific SCC3/SA paralog works
exclusively in homolog conjunction in meiosis I in male Drosophila but is not
required for sister chromatid cohesion in males or for any aspect of female meiosis
(Thomas et al., 2005; Soltani-Bejnood et al., 2007). In addition, a Rec8 subunit has
not been identified in Drosophila (Heidmann et al., 2004)
MEI-S332 and ORD are two meiotic cohesion proteins found in Drosophila so
far. Mei-S332 is the founding member of the Shugoshin family that protects
centromeric cohesion. Mei-S332 localizes to centromeres in both male and female
meiosis from PMI to MII, and dissociates concomitantly with segregation of sister
chromatids, which is consistent with its role in maintaining sister chromatid cohesion
(Kerrebrock et al., 1995; Moore et al., 1998). Several papers showed that mutations in
the mei-S332 gene cause premature separation of the sister chromatids in AI, resulting
in random sister chromatid segregation in meiosis II (Davis 1971; Goldstein et al.,
21
1980; Kerrebrock et al., 1992; Tang et al., 1998). These findings implied that
Mei-S332 functions in centromeric cohesion protection at AI, but the detailed
mechanism is unclear. Mei-S332 is also expressed in mitotic cells and localizes to the
centromeres from prometaphase to anaphase, however, unlike the mitotic shugoshins,
it is not required for mitotic cohesion, nor for centromere retention of Rad21 (Katis et
al., 2004; Kitajima et al., 2004; Lee et al., 2005). In addition, the centromeric
localization of Mei-S332 in mitosis and meiosis is directly regulated by the
chromosomal passenger complex, INCENP and Aurora B, and does not depend on the
cohesin complex (Lee et al., 2004; Resnick et al., 2006). Recently, Clarke et al. (2005)
showed that Mei-S332 activity is inhibited by POLO kinase and its removal from the
centromeric region is regulated by phosphorylation by POLO kinase (Clarke et al.,
2005; Lake et al., 2005).
ORD is a meiotic cohesion protein with no homologs in other organisms
(Bickel et al., 1996). ORD associates with the meiotic chromosomes during early PI
in male meiosis, but localizes to centromeres after chromosome condensation in PI
and persists until centromeric cohesin is released during AII (Balicky et al., 2002).
ORD functions in sister centromere orientation in meiosis I as well as in maintaining
sister chromatid cohesion in meiosis (Miyazaki et al., 1992; Bickel et al., 1997). ORD
also localizes to synaptonemal complexes in PI in females and is required for normal
recombination levels and for homologue bias during meiotic recombination.
Mutations in ord can cause premature desynapsis and reduced recombination. In
addition, Webber found that ORD is required for chiasmata maintenance in
22
Drosophila oocytes (Bickel et al., 1997; Bickel et al., 2002; Webber et al., 2004).
23
Chapter II – Evidence for a homolog
conjunction complex in Drosophila male meiosis
24
II-1 Introduction
Stromalin in Meiosis (SNM)
SNM is a protein which contains 973 amino acids and shares homology with
yeast SCC3, S. pombe REC11 and the vertebrate SA/STAG proteins, which are
components of cohesin and essential for sister chromatid cohesion (Prieto et al., 2001;
Kitajima et al., 2003). SNM is a meiosis-specific protein, but it does not function in
sister chromatid cohesion and is not orthologous to these proteins. It is a paralog of
Drosophila SA protein, which is thought to function in sister chromatid cohesion.
Other meiosis-specific SCC3/SA paralogs, such as S. Pombe Rec11 and vertebrate
STAG, have been described, but each of these proteins is more similar to its mitotic
paralog than to the other meiosis-specific paralogs, suggesting independent origins for
the meiotic paralogs (Thomas et al., 2005)
Modifier of Mdg4 in Meiosis (MNM)
MNM is a BTB-domain protein encoded by the mod (mdg4) locus, a complex
locus which encodes over 30 different proteins by alternative splicing and functions in
Drosophila in several processes, including chromatin boundary formation, nuclear
architecture, position effect variegation (PEV), apoptosis, regulation of homeotic
genes and early development, meiotic pairing of chromosomes and neurogenesis.
These Mod(mdg4) protein isoforms share a common N-terminal region of 402 amino
acids containing the BTB/POZ domain, a widely conserved protein-protein interaction
motif which is responsible for the formation of multimeric complexes or interaction
with other proteins. These isoforms vary in their C-terminal ends which contain from
25
28 to 208 amino acids. Most of the C-termini encode a Cys2His2–embedded
FLYWCH domain named by the six conserved hydrophobic amino acids (FLYWCH)
(Dorn et al., 1993; Gerasimova et al., 1995; Mackay J.P., et al., 1998; Gorczyca et al.,
1999; Buchner et al., 2000; Dorn et al.,, 2003, Thomas et al., 2005). In addition to
Drosophila, the FLYWCH motif is also present in three predicted mammalian
proteins and three additional C. elegans proteins of unknown function. The function
of the FLYWCH domain has not been proved, but Beaster-Jones found it is required
for the DNA binding and in vivo function of C. elegans PEB-1 (Beaster-Jones et al.,
2004). In addition, two Mod (Mdg4) isoforms, Mod(mdg4)-67.2 and
Mod(mdg4)-56.3/DOOM/MNM, indicate a role for this domain in protein-protein
interactions. Mod(mdg4)-67.2 interacts with the DNA binding protein Su(Hw) via its
specific C-terminus containing FLYWCH (Gause et al., 2001; Ghosh et al., 2001). For
the interaction between Mod(mdg4)-56.3/DOOM/MNM and the inhibitor of apoptosis
protein of Baculovirus OpIAP, the FLYWCH domain has been proved to be necessary
and sufficient (Harvey et al., 1997; Dorn et al., 2003).
The BTB domain (also known as the POZ domain (Poxvirus zinc finger)) in
the N-terminus of protein MNM was first identified by Koonin in 1992 as a conserved
sequence motif in genes of DNA virus and named from the Drosophila transcription
factors Bric-a-brac, Tramtrack, and Broad Complex which contains a similar
sequence at their N terminus. (Koonin et al., 1992; Godt et al., 1993; Zollman et
al.,1994). The BTB domain is a versatile protein-protein interaction motif and known
for its ability of dimerization, oligomerization and interaction with a number of other
26
BTB proteins or non-BTB proteins using its unique tri-dimensional fold with a large
interaction surface formed by approximately 95 core amino acids (Albagli et al., 1995;
Mazur et al., 2005). This unique character provides the BTB-containing proteins with
a variety of functional roles in transcription repression (Melnick et al., 2000; Ahmad
et al., 2003), cytoskeleton regulation (Ziegelbauer et al., 2001; Kang et al., 2004),
tetramerization and gating of ion channels (Kreusch 1998; Minor et al., 2000; )，
protein ubiquitination /degradation (Pintard et al., 2003; Furukawa et al., 2004;
Wilkins et al., 2004) and neurite outgrowth (Kim et al., 2005). The sequence of BTB
domains among BTB-proteins have been proved to be variable, though there are a
dozen highly conserved hydrophobic residues, and to form four known structural
classes with different solvent exposed surfaces, which is responsible for the different
oligomerization or protein-protein interaction states involving different
surface-exposed residues (Stogios et al., 2005). For instance, the T1 domains in ion
channel T1 proteins consist only of the core BTB fold without any amino- or
carboxy-terminal extensions and have a tendency of tetramerization, but Skp1
proteins contain the core BTB fold with two additional carboxyl-terminal helices
which provide another binding site (Kreusch et al., 1998; Bai et al., 1996). The other
two families are the BTB-zinc finger (BTB-ZF) family and ElonginC proteins. The
former can self-associate and dimerize because of an amino-terminal extension in
BTB domain, however the latter lacks the last alpha-helix of BTB domain which
affects its protein-protein interaction state (Ahmad et al., 1998; Botuyan et al., 2001).
Currently, BTB-protein families show more than two dozen different domains are
27
associated with one single copy of BTB domain, of which five are much more
frequent than others including MATH, Kelch, NPH3, Ion transport and Zinc finger
(ZF). The largest group is BTB-ZF proteins including the newly identified protein
MNM (Stogios et al., 2005; Perez-Torrado et al., 2006).
Two findings attract us to explore the interaction between proteins MNM and
SNM. One is their similar functions. Present evidence indicates that MNM and SNM
have similar functions in homolog conjunction. Mutations in the mnm and snm genes
result in homolog NDJ in meiosis I in male Drosophila. The second factor is their
appearance and localization. Both of them appear at the onset of PI and co-localize to
the XY homologous pairing site at MI, then disappear suddenly at AI (Thomas et al.,
2005). Are they co-partners or different components of s homologous conjunction
complex in male Drosophila?
28
II-2 Materials and Methods
E.coli strains, Yeast Strains and Plasmids
The Chemically Competent E. coli { F- mcrA Δ(mrr-hsdRMS-mcrBC)
φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(araleu) 7697 galU galK rpsL (StrR)
endA1 nupG} was purchased from Invitrogen company and transformation of E.coli
were performed according to its protocol: cells on LB agar plates or liquid with
100 µg/ml ampicillin were incubated at 37 °C over night in the incubator (REVCO)
or with shaking at 220 r.p.m. respectively. Yeast strain PJ69-4A (MATa trp1-901
leu2-3,112 ura3-52 his3-200 gal4
gal80
LYS2::GAL1–HIS3 GAL2–ADE2
met2::GAL7–lacZ; James et al., 1996) was a gift from Dr. Guan. PJ69-4A contains
three separate reporter genes under the independent control of three different GAL4
promoters respectively (GAL1-HIS3, GAL2- ADE2 and GAL -lacZ) and provides a
high level of sensitivity with respect to detecting weak interactions, coupled with a
low background of false positives (James et al., 1996). The plasmids, pGAD-C1 and
pGBD-C1 (Fig II-1), were gifts also from Dr. Guan and their structures are shown in
the follow figure. The vector pGAD-C has a selective gene leu2 to ensure that yeast
colony containing the corresponding plasmid or plasmid construct can grow up on
plates minus leucine. The vector pGBD-C has a selective gene trp1 to select the yeast
colony which contains the plasmid or plasmid construct on plates minus tryptophan.
Plasmid Construction and DNA Sequencing
The mnm cDNA was PCR amplified using the sense primer with an EcoRI site
(5’-GATCGAATTCATGGCGGACGACGAG-3’) and the anti-sense primer with a
29
Figure II-1. The structures of the pGAD-C and pGBD-C vectors. Stippled
regions indicate the ADHl promoter (P) and transcription termination (T) elements.
GAL4 AD (activation domain) in pGAD-C encodes amino acids (768-881). GAL4
BD (DNA binding domain) in pGBD-C encodes amino acids (1-147). Restriction sites
following by GAL4 AD or GAL4 BD are shown on each map including EcoRI, SmaI,
BamHI, ClaI, SalI, PstI and BglII. Both vectors contain the amp gene to allow the
E.coli selection on the LB plate added with ampicillin.
30
BamHI site (5´-GATCGGATCCCTACAAATGGTTGTGC-3´). The gene snm cDNA
was amplified using the forward primer with a BamHI site
(5’-CAGCTTGGATCCATGAGTGATATATCTTTTGATG-3’) and the reverse primer
with a SalI site (5´-GTAGCGTCGACCATCCTGTAAGTTGTATCCTTC -3´).
Full-length snm and mnm cDNA templates were described in Thomas et al., 2005 and
were kindly provided by Dr. S. Thomas. The btb cDNA was amplified by the sense
primer with an EcoRI site (5’-GATCGAATTCATGGCGGACGACGAG-3’) and the
anti-sense primer with a BamHI site
(5´-GATCGGATCCCTACAAATGGTTGTGC-3´).This btb product contains 127
amino acids of the N-terminus of MNM protein (Fig II-2). The mnm PCR product
with both vectors were digested with both EcoRI and BamHI (Invitrogen) and were
ligated separately by T4 DNA ligase (Invitrogen) in according with the provided
protocols to construct pGAD-MNM and pGBD-MNM, simply AD-MNM (AM) and
BD-MNM (BM). The same process was performed on the snm and btb PCR products
to constitute AD-SNM (AS), BD-SNM (BS), AD-BTB (AT) and BD-BTB (BT). After
confirmation by DNA sequencing, these constructs were transformed into E.coli (FmcrA
(mrr-hsdRMS-mcrBC)
80lacZ M15
lacX74 recA1 ara 139
(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG) provided by Invitrogen.
Colony PCR and Plasmid Extraction
The optimized colony PCR reaction mixture contained 1X PCR amplification
buffer [20 mM (NH4)2SO4, 72.5 mM Tris/HCl, 0.1% Tween 20, pH 9·0], 2.5 mM
31
Figure II-2. Structure of BTB domain and FLYWCH domain of MNM protein.
32
MgCl2, 200 µM each deoxynucleotide triphosphate, 2.5 µM each primer, 1.25 U
Supertherm DNA polymerase (LPI) in 50 µl PCR reaction mixture. A final
concentration of 100 µg / ml of acetylated BSA (New England BioLabs), 3%
dimethyl sulfoxide (DMSO) (Sigma) and 1 M betaine (Sigma) as PCR additives were
also added to the reaction mixture. Colonies approximately 1 mm in diameter were
picked up with a sterilized toothpick and directly transferred to the PCR tube as DNA
templates. The thermal cycle program, run on a GeneAmp PCR system 9700 (Perkin
Elmer) consisted of one cycle of 94 °C for 10 min, 51 °C for 2 min, 72 °C for 2 min,
and 35 cycles of 94 °C for 20 s, 57 °C for 45 s (decreased by 1 s per cycle), 72 °C for
1 min, and then incubation at 72 °C for 5 min, and a final incubation at 4 °C. (Sheu et
al., 2000). After amplification, a PCR product was subjected to gel electrophoresis
analysis and DNA sequence analysis to select the plasmid that contains recombinants
of the vector and the protein of interest without any mutation.
Yeast Two-Hybrid Assay and β-galactosidase Assay
Two-hybrid assays were performed using yeast strain PJ694A and plasmids
provided by Dr. Guan. For growth assays, plasmids were transformed into yeast strain
PJ694A by the lithium acetate method described in the yeast protocols handbook
(Clontech). Bait and target fusion proteins were produced constitutively under the
control of the ADH1 promoter. Co-transformants were plated on selective media
minus tryptophan and leucine. After incubation at 30°C for 3 days, plates were
replicated on selective media lacking tryptophan, leucine and histidine or selective
33
media lacking tryptophan, leucine and adenine (selective plates for the reporter gene),
and growth on both plates was compared. Appearance of transformants on the
selective plates indicates a positive interaction. Single colonies were subsequently
streaked out on selective plates to obtain the plates shown in the following figures.
β-Galactosidase activity was determined for liquid cultures grown in
yeast-peptone-dextrose medium by using the ONPG
(O-nitrophenyl-b-D-galactopyranoside) assay. One yeast colony from each section on
the media without leucine and tryptophan was picked and shook (230 rpm) overnight
in 5ml YPD liquid at 30℃. Cells were suspended in breakage buffer (0.1 M Tris pH
8.0, 1 mM DTT, 20% glycerol ) containing 1 mM PMSF (phenylmethane sulfonyl
fluoride, Sigma). Glass beads were added to disrupt the cells and the suspensions
were cleared by centrifugation after disrupting cells with glass beads. Protein
concentration was measured, and 300 ug of protein were used to perform the β-gal
activity assays in the Z buffer containing 200 ul of ONPG (ortho nitrophenyl
galactoside, Sigma) 4mg/ml. The final volume of each reaction was 500 ul. Finally,
the reactions were incubated at 32℃ until the solution became faint yellow and were
stopped by adding 0.5 ml of 1 M sodium carbonate. The OD420 of each sample was
measured and the β-gal units were calculated.
Statistics Analysis
All data were evaluated for normality of distribution and equality of variance
prior to statistical analysis. Outcomes were shown as means ± SD and were evaluated
34
for statistical significance by one-way analysis of variance (ANOVA) and t test to
compare group means using SPSS 14.0 (SPSS Inc, Chicago, IL).
35
II-3 Results and Discussion
1. MNM can interact with SNM and with itself
The finding that two proteins MNM and SNM co-localize to the X-Y
bivalents from late G2 through PMI and MI and disappear simultaneously at Ana I,
and that mutations in both mnm and snm show high similar meiotic phenotype,
suggests a hypothesis that these two proteins interact directly with each other. We
used the yeast two-hybrid system to test this hypothesis under in vivo conditions. We
cloned the full-length coding sequence of MNM and SNM, and that of the BTB
domain of MNM, into both the pGAD-C1 (yeast Gal4 activation domain) and
pGBD-C1 (yeast Gal4 DNA binding domain) yeast two-hybrid vectors. Here
BTB-only clones served as a positive control because the BTB domain is a versatile
protein-interacting motif and the BTB domain of Mod(mdg4) has been proved to
interact directly and strongly with itself (Albagli et al., 1995; Mazur et al., 2005).
These clones were then co-transformed into PJ69-4A yeast cells which contain three
independent reporter genes (GAL1-HIS3, GAL2-ADE2 and GAL7-lacZ). If two
proteins can interact with each other, the reconstituted intact Gal4, a transcription
regulating protein, has the ability to activate transcription of these reporter genes, and
allow growth of colonies. As expected, the colonies carrying BTB-only AD+DB
combination can grow on the media minus adenine (Fig.II-3-A) and media minus
histidine (Fig.II-3-B), indicating that the BTB domain can interact with itself and can
be used as a positive control. Also, the colonies carrying MNM-only AD+DB
combination also can grow on the media minus adenine (Fig. II-3-A) and the media
36
Figure II-3. MNM and SNM interact directly and weakly with each other. Growth of
yeast strain PJ69-4A expressing different proteins BTB, MNM, and /or SNM pairs on
non-selective (left) or selective (right) media for the reporter gene HIS3 (A) and ADE
(B) used in yeast two-hybrid system. The numbers on the plate indicate the following:
1, yeast expressing AD-BTB+BD-BTB; 2, yeast expressing AD-MNM+BD-MNM; 3,
yeast expressing AD-SNM+BD-MNM; 4, yeast expressing AD-MNM+BD-SNM; 5,
yeast expressing AD-MNM+BD; 6, yeast expressing AD+BD-MNM
The yeast colonies carrying both BTB-only AD+DB combination and
MNM-only AD+DB combination also can grow on the media minus histidine and the
media minus adenine, indicating that MNM can interact with itself.
37
2
2
3
3
1
1
4
4
6
6
5
5
Figure II-3-A
2
2
3
3
1
1
4
4
6
6
5
Figure II-3-B
38
5
without histidine (Fig.II-3-B), indicating that MNM, as a BTB-containing protein, can
interact with itself. Yeast co-transformed with SNM-BD and MNM-AD or with
SNM-AD and MNM-BD grew well on the media without histidine (Fig. II-3-B) but
poorly on the media without adenine (Fig.II-3-A). HIS3 is not ideal reporter because
of its leaky expression to yield high false positive frequency, but it is still useful for
the effective detection of weak interactions (James et al., 1996). Notably, both
SMNM-MNM combinations (section 3 and 4) exhibited much better growth on hisplates than did either MNM-BD +AD (section 5) and MNM-AD + BD (section6),
indicating that the growth in the SNM-MNM combination is not due only to leaky
HIS3 expression. Thus, these data suggest that MNM-SNM interact with each other.
The SNM-MNM interaction was further confirmed in liquid culture assays of
β-galactosidase activity, which is the third reporter gene of the system. Yeast
co-transformed with MNM-only AD+DB combination, SNM-BD and MNM-AD , or
SNM-AD and MNM-BD produced similar levels of β-gal, which are significantly
higher than when expressing either MNM-BD or MNM-AD alone (p<0.05) (Fig.II-4).
Although this expression is lower than theβ-gal units produced by the BTB-BTB
interaction, we still can draw the conclusions that MNM protein interacts with itself,
and that MNM and SNM proteins can interact with each other.
In addition, yeast which expresses AD+BD-MNM was also found to grow up
on the media without histidine. We think that it is the consequence of the leaky
expression of histidine because its β-gal unit is much lower than the other positive
combinations and similar to the other negative control which contains AD-MNM+BD.
39
β - gal Uni t
250
*
200
150
100
**
**
50
0
AT+BT
AM+BM
**
AM+BS
AS+BM
***
***
AM+BD
AD+BM
Figure II-4. β-galactosidase activity, expressed as Milller units, in extracts of yeast
strains carrying combinations of BTB, MNM, and /or SNM protein pairs. From left to
right: 1, yeast carrying AD-BTB+BD-BTB; 2, yeast carrying AD-MNM+BD-MNM;
3, yeast carrying AD-MNM+BD-SNM; 4, yeast carrying AD-SNM+BD-MNM; 5,
yeast carrying AD-MNM+BD; 6, yeast carrying AD+BD-MNM
* Value is significantly different from ** value by One-way ANOVA (p<0.05)
* Value is significantly different from *** value by One-way ANOVA (p<0.05)
** Value is significantly different from *** value by One-way ANOVA (p<0.05)
40
2. What part of MNM is responsible for the interaction between MNM and
SNM?
BTB domains or other BTB-ZF proteins have been shown to interact with
other proteins. Is the MNM BTB domain also involved in the interaction between
MNM and SNM proteins? In support of this, we found that yeast co-transformed with
SNM-BD and BTB-AD or with SNM-AD and BTB-BD grew vigorously on the
media without histidine, suggesting that the BTB domain alone interacts with SNM
protein (Fig.II-5). However, theβ-gal units produced by yeast co-transformed with
SNM and BTB were much lower than those produced by yeast expressing SNM and
MNM (Fig. II-6). This implies that another part of MNM protein is involved in the
interaction between MNM and SNM.
3. Unanswered questions and future experiments
Although BTB-only interacted with SNM to activate both HIS3 and lacZ reporter
genes, full-length MNM interacted more strongly with SNM than did BTB-only, at
least in the β-gal assay, suggesting that an additional domain of MNM outside the
BTB domain may contribute to the SNM-MNM interaction. Previous research proved
that the FLYWCH domains in the C-termini of proteins Mod (mdg4)-67.2 and Mod
(mdg4)-56.3/DOOM/MNM have roles in specific protein-protein interactions. They
can interact independently with Su(Hw) and OpIAP respectively (Gause et al., 2001;
Ghosh et al., 2001). Therefore, the FLYWCH domain of MNM is a good candidate for
the second SNM-MNM interaction domain. An experiment is in progress to determine
41
3
2
2
3
4
4
1
1
5
5
7
7
6
6
Figure II-5. Growth of yeast strain PJ69-4A expressing different proteins BTB, MNM,
and /or SNM pairs on non-selective (left) or selective (right) media for the reporter
gene HIS3 used in yeast two-hybrid system. The numbers on the plated denote the
following:
1, yeast expressing AD-BTB+BD-BTB; 2, yeast expressing AD-BTB+BD-MNM;
3, yeast expressing AD-MNM+BD-BTB; 4, yeast expressing AD-BTB+BD-SNM;
5, yeast expressing AD-SNM+BD-BTB; 6, yeast expressing AD-BTB+BD;
7, yeast expressing AD +BD-BTB.
42
90
80
70
60
50
40
30
20
10
0
66. 85
49. 55
MNM & SNM
BTB & SNM
19. 425
9. 725
1
2
Figure II-6. β-galactosidase activity, expressed as Milller units. Comparison of β-gal
units produced by the yeast colony containing MNM and SNM proteins and by the
yeast colony carrying BTB domain and SNM protein.
1: β-gal units produced by AM+BS (blue) and AT+BS (red)
2: β-gal units produced by AS+BM (blue) and AS+BT (red)
43
the interaction between the FLYWCH domain of MNM and SNM by using the
isolated C-terminus of MNM. If the isolated FLYWCH domain is able to interact with
SNM, a useful follow-up experiment would be to determine whether this interaction
depends on the C2H2 motif because a single amino acid substitution in the C2H2 motif
that changes it to CYCH was found to completely abolish MNM meiotic function and
disrupt its location to the nucleolus and to chromosomes (Thomas et al.,2005;
Soltani-Bejnood et al., 2007). Thus, it would be informative to introduce the CYCH
mutation into both full length MNM and FLYWCH only MNM to determine whether
the C2H2 motif is required for the interaction with SNM.
In all of the experiments reported above, the Drosophila proteins were fused at
their N-termini to the C-terminus of either Gal4-BD or Gal4-AD. Mazur found in
2005 that when GAL4 activation domain (AD) is fused with the N-termini of the BTB
domain or of a BTB-containing protein, its ability to induce transcription is disturbed,
resulting in difficulty in detecting the interaction between proteins using the Yeast
Two-Hybrid Assay (Mazur et al., 2005). So, an alternative experiment in the future is
to switch the BTB and MNM protein from the C-termini of AD to the N-termini of
AD to measure the interaction strength between BTB and SNM or between MNM and
SNM.
44
Chapter III – sun is required for sister
chromatid cohesion and sister centromere
mono-orientation in male Drosophila meiosis
45
III – 1 Introduction
Recently, S. Thomas and B. McKee have identified a new Drosophila gene,
sisters unbound (sun) which was discovered in a genetic screen for EMS-induced
mutations that disrupt paternal transmission of the small 4th chromosome (Wakimoto
B.T. et al., 2004), and mapped to the 68D3 region on the left arm of 3rd chromosome.
All four sun alleles have mutations in the predicted coding sequence of CG32088, a
gene predicted to consist of 9 exons and to encode a protein 760 amino acids in length
(Fig III-1). sun is required for centromeric cohesion. Mutations in sun cause abnormal
meiosis in both male and female Drosophila including high homologous and sister
chromatid NDJ, resulting in aneuploid sperm. sun mutations also affect recombination
and synapsis in females meiosis. My study focused on the role of sun in centromeric
cohesion. How and when do sun mutations affect cohesion and sister chromatid
segregation in meiosis in male Drosophila? During which phase is centromeric
cohesion first lost, PI or MI? The proteins SNM and SUN interact in sister chromatid
cohesion in male Drosophila.
46
CG32088/sun
Z3-5839
Del. Stop
5’
1
3’
2
3
4
6
5
7
8
Z3-4085
Z3-1550
Z3-1956
G170R Missense
Q557 Stop
W839R Stop
9
Figure III-1. Molecular characterization of CG32088/sun. All alleles are shown above
and below the gene model. Grey shading represents the exons.
47
III- 2 Material and Methods
Fly stocks, Special Chromosomes and Drosophila Culture Methods.
The sun mutations in this study were from the Zuker-3 (Z3) collection of more
than 6000 lines with EMS-mutagenized third chromosomes which was produced in a
screen for paternal 4th chromosome loss (Koundakjian et al., 2004; Wakimoto et al.,
2004), and were provided kindly by B. Wakimoto. All flies were maintained at 23°C.
Compound chromosomes and markers are described in Flybase. CID-GFP stocks
were furnished kindly by S. Henikoff. Unless otherwise noted, tested males were
crossed singly to two or three females in shell vials. Crosses were incubated at 23◦C
on cornmeal-molasses-yeast-agar medium. Parents were removed from the vial on
day 10 and progeny were counted between day 13 and day 21.
Sex Chromosome Nondisjunction Test in males
Sex
chromosome
nondisjunction
was
measured
in
crosses
of
trans-heterozygous males for two sun alleles (sunZ3-5839/sunZ3-1956) having a marked Y
chromosome to the females carrying the compound-X chromosomes, C(1)RM. These
female can produce only nullo-X and diplo-X chromosomes eggs equally. The
nullo-X eggs generate viable progeny when fertilized by either XX or XY sperm, the
sperm classes that are diagnostic for sister chromatid and homolog NDJ, respectively,
and the diplo-X eggs generate viable progeny when fertilized by nullo-XY (O) sperm.
The cross can also yield progeny from XXY, XYY and XXYY sperm, but such
progeny were recovered only very rarely and were neglected in this analysis.
48
Testis Immunostaining and FISH
For anti-α-tubulin/DAPI experiments, testes were fixed according to Cenci et
al. (1994) and stained according to protocol 5.6 (Bonaccorsi et al., 2000).
FITC-conjugated monoclonal anti-α-tubulin (Sigma) was diluted 1:150. (Thomas et
al., 2005).
CID-GFP Detection in Spermatocytes
For CID-GFP experiments, testes were dissected from young adults in testes
buffer (183 mM KCl, 47 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, 1 mM PMSF),
gently squashed and frozen in liquid nitrogen. Slides were then incubated with 1
mg/ml 4'-6-Diamidino-2-phenylindole (DAPI) at room temperature for 10 minutes
and washed with 1XBBS twice for five minutes.
Microscopy and Image Processing
All images were collected using an Axioplan (ZEISS) microscope equipped
with an HBO 100-W mercury lamp and high-resolution CCD camera (Roper). Image
data were collected and merged using Metamorph Software (Universal Imaging
Corporation). For CID signals, all images were taken as Z-series by using a 100×
oil-immersion objective, and were deconvolved by using Metamorph Software to
obtain maximum projections.
49
III- 3 Results
1. NDJ of homologous and sister chromatids in sun males
To measure whether sun mutations disrupt segregation of the sex
chromosomes in Drosophila male meiosis, sun males (sunZ3-5839/sunZ3-1956) carrying a
dominantly marked Y chromosome were crossed to C(1)RM females which carry the
attached-X chromosome and can generate either nullo-X or diplo-X eggs at equal
ratio. After the cross, we confirmed that mutations in sun cause both homolog and
sister chromatid NDJ, resulting in XX and XY sperm classes (Fig. III-2). Assuming
that YY sperm are produced at frequencies comparable to those of XX sperm, the
ratio of sister chromatid to homolog NDJ (S/H) is 0.70, which indicates that the four
sex chromatids separate prematurely during meiosis I and segregate approximately
randomly through both divisions.
2. Sister centromere cohesion is lost at mid-prophase in sun spermatocytes
Surprisingly, in DAPI-stained preparations we found that chromosome
morphology in meiosis I in sun mutants is similar to that in wild-type. Bivalents were
nearly always intact during PMI and MI and segregated evenly to opposite poles (Fig.
III-3), although it was often seen that sister chromatids segregate unevenly in MII
(Fig.III-4). One possible explanation here is that SNM & MNM hold bivalents
together and prevents the complete separation of homologous and sister chromatids in
sun mutants in meiosis I. In order to assess the effects of sun mutations on
centromeric cohesion and to determine in which stage centromeric cohesion is lost in
50
Test of homol og vs si st er chr omat i d NDJ f or sun
mut ant s
300
250
244
200
200
176
150
99
100
35
50
0
X
Figure III-2.
Y
XX
XY
0
NDJ test of homologs vs sister chromatids for sun mutants. The
X-coordinate represents different possible sperm classes and the Y-coordinate is the
number of F1 male Drosophila. The S/H ratio {(XX+YY)/XY} here was 0.70 and the
overall NDJ frequency was 0.44.
51
PMI
MI
AI
TI
Wild Type
sun
Figure III-3. Chromosome morphology is normal during meiosis I in sun
spermatocytes (sunZ3-5839/sunZ3-1956), compared to wild-type. Bivalents were nearly
always intact during PMI and MI and segregated evenly to opposite poles in AI and
TI. Red-DAPI, Green-α-tubulin.
52
PMII
MII
AII
TII
Wild Type
sun
Figure III-4. Sister chromatids segregate unevenly in meiosis II in sun spermatocytes
(sunZ3-5839/sunZ3-1956). Red-DAPI, Green-α-tubulin.
53
meiosis I, we generated trans-heterozygous sun males (sunZ3-5839/sunZ3-1956) which
express a CID-GFP fusion protein. CID (Centromere identifier) is the Drosophila
homolog of the CENP-A centromere-specific H3-like proteins and localizes
exclusively to the centromeres (Ahmad et al., 2001; Blower et al., 2001). In our study
we used CID to diagnose the sister centromere’s cohesion status in meiosis in
wild-type or sun spermatocytes.
In wild-type spermatocytes, sister centromeres are tightly held together
throughout meiosis I because of centromeric cohesion, so there are never more than
eight separate CID spots, corresponding to the eight chromosomes in a diploid cell.
As expected, sun mutants exhibited too many CID spots in mid-prophase (S4) stage
(Fig. III-7). Most bivalents in sun spermatocytes exhibited 3 or 4 spots instead of the
normal 2 spots and the total spot number per nucleus was more than 8, indicating a
defect in centromeric cohesion. Similar results were obtained with mature
spermatocytes in stages S5 (Fig. III-8). However, during early-(S1/2) and
mid-prophase (S3) the numbers of CID foci in sun mutants were similar to wild-type
(Fig. III-5 and III-6) (Cenci et al., 1994). We concludethat in sun mutants centromere
cohesion becomes lost after resolution of chromosome territories in mid-prophase
(S4). These observations strongly suggest that the aberrant meiosis I segregation
patterns apparent in the cross data result from premature loss of centromeric cohesion,
allowing sister chromatids to segregate nearly randomly.
54
DNA
CID
MERGE
S1/2
Wild Type
sun
Figure III-5. Less than 4 CID-GFP spots are frequent in both sun (sunZ3-5839/sunZ3-1956)
and wild-type spermatocytes in early-prophase I (S1/2). Each circle represents one
cell. Green – CID-GFP; Red – DAPI.
55
DNA
CID
MERGE
S3
Wild Type
sun
Figure III-6. In mid-prophase I (S3) both sun (sunZ3-5839/sunZ3-1956) and wild-type
spermatocytes showed less than 4 CID-GFP spots. Each circle represents one cell.
Green – CID-GFP; Red – DAPI.
56
DNA
CID
MERGE
Wild Type
sun
Figure III-7. Centromeric cohesion is lost at stages S4 in sun spermatocytes
(sunZ3-5839/sunZ3-1956). Green – CID-GFP; Red – DAPI.
57
S4
DNA
CID
MERGE
S5
Wild Type
sun
Figure III-8. Centromeric cohesin is absent during stages S5 in sun spermatocytes
(sunZ3-5839/sunZ3-1956). Green – CID-GFP; Red – DAPI. It is more clear that the sun
spermatocyte exhibits more than 8 CID-GFP spots in late-prophase I.
58
III-4 Discussion
sun is required for centromere cohesion
Our data showed that SUN is required for sister centromere cohesion in male
Drosophila meiosis. Mutations in sun cause loss of sister centromere cohesion as
early as mid-prophase (S4), although bivalents remain intact through meiosis I.
Homologous and sister chromatids segregate approximately randomly and yield
aneuploid sperm.
The recovery of sperm containing both X and Y chromosomes indicates that
mono-orientation of sister centromeres during meiosis I is disrupted in sun mutations.
However it is not clear whether this disruption is a consequence of loss of sister
centromere cohesion or represents another function for SUN. There is evidence that
cohesion between sister centromeres is required for their “mono-orientation” to the
same pole on the meiosis I spindle, although some new proteins also have been
proved to be required for sister centromere mono-orientation, such as Monopolin in S.
cerevisiae and Moa1 in S. pombe (Toth et al., 2000; Yokobashi et al., 2004). For
example, mutations in the meiosis-specific cohesin subunit Rec8 result in bipolar
attachment of sister kinetochores in meiosis I (Watanabe 1999; Yokobayashi et al.,
2003; Yamamoto et al., 2003; Hauf et al., 2004). So, it is important to determine the
real function of SUN in mono-orientation of sister centromeres.
Is SUN a component of cohesin complex?
Although SUN is required for sister centromere cohesion, we do not know
59
whether SUN is a component of cohesin complex or it works as the protector for
sister centromere cohesion. In S. cerevisiae and S. pombe, meiotic cohesion functions
are fulfilled by cohesin complexes that include meiosis-specific subunits such as
REC8, which replaces the mitotic kleisin subunit RAD21. Rec8 conserved among
most eukaryotes, but no true REC8 homolog has been identified in Drosophila,
(Petronczki et al., 2003; Heidmann et al., 2004; Losada et al., 2005). In addition, no
mutations in cohesin genes have been available in Drosophila. Thus the role of
cohesin in meiotic cohesion is unclear. The Drosophila ORD is a meiotic cohesion
protein with no homologs in other organisms. Protein ORD localizes to centromeres
after chromosome condensation in PI and is required in meiosis for both arm and
centromere cohesion (Bickel et al. 1997; Balicky et al. 2002). The phenotypes of sun
mutations are similar to those of ord mutations in Drosophila. Both sun and ord
mutations cause premature loss of centromere cohesion during meiosis I, leading to
NDJ of both homologous and sister chromatids (Miyazaki et al. 1992; Bickel et al.
1997). So, it is interesting to uncover the relationship between sun and ord. Does SUN
also localize to centromeres? Do they work together as partners, or is SUN a protector
of ORD?
In addition, proteins Shugoshin (Sgo1) and PP2A phosphatase have been
proved to work together to shield centromeric cohesin in yeast (Kitajima et al., 2006;
Riedel et al., 2006). As the founding member of the Shugoshin family, Drosophila
MEI-S332 is also a meiotic cohesion protein and protects centromeric cohesion.
Mutations in the mei-S332 cause premature separation of the sister chromatids in
60
meiosis II. It is another direction for us to discover the real function of SUN in
centromere cohesion and the relationship between SUN and MEI-S332.
61
Chapter IV – Roles of interaction of sun, snm
and mnm in meiotic cohesion, centromere
pairing and chromosome territory integrity
62
IV-1 Introduction
Crossovers are important for homologous chromosomes segregation during
meiosis I. However, in several organisms, such as male Drosophila, it has been shown
that nonexchange pair of chromosome also can be segregated properly, which
demonstrates that some unknown mechanisms, other than exchange, are involved in
the nonexchange segregation. Studies in Drosophila females and S. cerevisiae have
shown that pairing at the centromeric region is required for correct homologous
chromosome segregation when chiasmata are absent. In S.cerevisiae centromere
regions undergo meiotic pairing which is sequencing-independent, and this pairing
orients the kinetochore of the nonexchange partners (Karpen et al., 1996; Kemp et al.,
2004). Another finding also using S. cerevisiae showed that initially centromeric
interactions occur mainly between non-homologous chromosome centromeres, and
then switch to homologous centromeres prior to zygotene. These centromeric
interactions are dependent on a component of the SC, Zip1, and the transition from
non-homologous to homologous centromeres pairing is dependent on Spo11, the
endonuclease required for DSB formation during meiosis (Tsubouchi et al., 2005).
Nsl1p, a new protein important for chromosome segregation, has also been found to
play a role in transient centromere pairing in S.cerevisiae (Nekrasov et al., 2002).
However in male Drosophila, it is not clear about homologous centromeres pairing. In
our research, we found that SNM protein play a roles in homologous centromeres
pairing in the stage S3 in PI, and loss of SNM causes homologous centromere
unpaired.
63
IV-2 Materials and Methods
All materials and methods in this chapter are similar to that in the chapter III.
In this chapter trans-heterozygous males for two snm alleles (snmZ3-2138/snmZ3-0317)
and for two mnm alleles (mnmZ3-5578/mnmZ3-3401) were used.
64
IV-3 Results
1. The homolog conjunction protein SNM prevent complete separation of
homologous and sister chromatids in sun mutants
To test the possibility that the homolog conjunction proteins can hold sister
chromatids together in the absence of SUN, males doubly mutant for sun snm
(sunZ3-5839 sunZ3-1956 / snmZ3-2138 snmZ3-0317) were generated and their chromosomes were
examined by DAPI staining. SNM is a homolog conjunction protein in male
Drosophila and plays a role in holding homologous together from PI to AI. Loss of
SNM will cause homolog NDJ in meiosis I (Thomas et al., 2005). As shown in Figure
IV-1, the double mutants displayed a much more severe phenotype during meiosis I
than single sun mutants, or single snm mutants, and fully separated sister chromatids
can be seen during PMI and MI.
In addition, the theoretical S/H (sister/homolog) NDJ ratio of random 2x2
segregation at meiosis I followed by random segregation at meiosis II should be 0.5,
whereas the observed ratio in single sun mutants is 0.70, which implies that the
homolog conjunction protein promotes reductional (XX:YY) segregations at meiosis I.
To test this possibility, sun and snm males carrying BSYy+ were crossed with
C(1)RM/O females (Table IV-2). The S/H ratio was reduced to 0.53, close to 0.50,
confirming that sister chromatids segregate completely randomly in double mutants
males. So we concluded that the homolog conjunction protein SNM prevents
complete separation of homologous and sister chromatids in sun mutants. Although
SNM alone does not suffice to maintain sister chromatid mono-orientation, it does
65
Wild type
sun
snm
sun & snm
PMI
MI
Figure IV-1. Sister chromatids segregate completely during PMI and MI in sun snm
double mutants (sunZ3-5839/sunZ3-1956 snmZ3-2138/snmZ3-0317) Red-DAPI, Green-α-tubulin
66
mutants
X
Y
XX
YY
XY
O
244
200
35
35
99
176
sun
30.9%
25.3%
4.4%
4.4%
12.5%
22.3%
691
546
57
57
217
953
sunsnm
27.4%
21.6%
2.3%
2.3%
8.6%
%NDJ
S/H
44%
0.70
51%
0.53
37.8%
Table IV-2. The percentage comparison of different sperm classes produced by single
sun (sunZ3-5839/sunZ3-1956) mutants and sun snm (sunZ3-5839 sunZ3-1956 / snmZ3-2138 snmZ3-0317)
double mutants males. The S/H ratio {(XX+YY)/XY} in single sun mutants was 0.70,
and in sun snm double mutants was 0.53 which is close to 0.50, confirming that sister
chromatids segregate completely randomly in double mutants males.
67
promote a non-random excess of reductional segregation.
2. SNM, but not MNM, is required for the pairing of homologous centromeres
during mid-prophase (S3)
Vazquez et al. reported in 2002 that homologous centromeres in wild type
males can pair transiently during stage S3, but suddenly lose pairing by stage S4
(Vazquez et al., 2002). To determine whether the homolog conjunction complex plays
a role in the centromeric pairing, both snm and mnm males expressing CID-GFP were
generated. In wild-type males and mnm males, each newly formed chromosome
territory shows a single CID-GFP spot in S3 (no more than 4 CID-GFP spots can be
seen in one nucleus), and eight CID spots from stage S4 through MI (data not shown).
However, in snm males, more than 99% of S3 nuclei exhibited five or more CID-GFP
spots, and most exhibited 6-8 spots (Fig.IV-3). Thus, we concluded that SNM, but not
MNM, is required for the pairing of homologous centromeres in the stage S3, and this
pairing is transient and is lost between stages S3 and S4.
Remarkably, sister centromeres separated earlier in PI in sun snm double
mutants than in either single mutants. In sun snm males there were more than 8
CID-GFP spots in 30% of S3 nuclei, 95% of S4, and 100 % of S5/6. However, in
single sun males, S3 nuclei exhibited normal frequencies CID-GFP spots. After that
42% of S4 and 90% of S5/6 nuclei showed more than 8 CID-GFP spots. This
indicated that SNM can maintain cohesion between sister centromeres in the absence
of SUN in the stage S3 (Fig. IV-4).
68
CID
DNA
MERGE
Wild Type
sun
snm
snm & sun
Figure IV-3. Sister centromere cohesion is lost in sun snm double mutants
(sunZ3-5839/sunZ3-1956 snmZ3-2138/snmZ3-0317)
in S3 stage, which is earlier than sun
single mutants, indicating that SNM can maintain cohesion between sister
centromeres in the absence of SUN in mid-prophase (S3). Red-DAPI,
Green-CID-GFP
69
CID spots in prophase I
> 8 CID spots percentage
120%
100%
95%
100%
90%
80%
WT
snm
sun
snm sun
60%
42%
40%
30%
20%
0%
0%
S1/ 2
0%
S3/ 4
S4
0%
0%
S5/ 6
Prophase I
Figure IV-4. A comparison of > 8 CID spots percentage in different stages in prophase
among
wild-type,
single
sun
(sunZ3-5839/sunZ3-1956)
mutants,
single
snm
(snmZ3-2138/snmZ3-0317) mutants, and sun snm (sunZ3-5839/sunZ3-1956 snmZ3-2138/snmZ3-0317)
double mutants.
70
3. Mutations in sun snm and sun mnm impair the integrity of chromosome
territories
Beginning at stage S3 (mid-G2) in wild type males the chromatin masses form
three main territories, presumably corresponding to the three major bivalents: XY, 2nd,
and 3rd chromosomes (Vazquez et al., 2002), and remain separate until the onset of MI.
In sun mutants, although centromeric cohesion is impaired and sister centromeres
separate prematurely, chromosome morphology and behavior during PI appear normal.
Three separate DAPI-stained masses, corresponding to the three major bivalents, were
regularly seen. In single snm mutants territory formation at stage S2b/S3 appeared
normal but in stage S4-S6, approximate 50% of snm spermatocytes exhibited unusual
diffuse territory (Thomas et al., 2005). Surprisingly, sun snm and sun mnm double
mutants severely disrupted the integrity of chromosome territories. Nearly all
territories during late PI were subdivided into smaller subterritories and exhibited
more than 8 chromatin clumps, each of which, we think, corresponds to a single
chromatid from a large chromosome (Fig. IV-5).
We conclude that the integrity of chromosome territories is dependent solely
and jointly on proteins that regulate homolog pairing and sister chromatid cohesion.
71
Wild Type
mnm & sun
snm & sun
Figure IV-5: The integrity of chromosome territories in late PI are impaired in
mutations in sun snm (sunZ3-5839/sunZ3-1956 snmZ3-2138/snmZ3-0317) and sun mnm double
mutants (sunZ3-5839/sunZ3-1956 mnmZ3-5578/mnmZ3-3401). Red-DAPI, Green-α-tubulin.
72
IV-4 Discussion
1. What does SNM do in centromere pairing and sister chromatid cohesion?
What is the role of homologous centromeres pairing in meiosis? One
explanation here is that homologous centromeres pairing are prerequisite to
homologous chromosomes pair and proper segregation during meiosis I. However,
little is known about centromere pairing. Tsubouchi et al., proposed in 2005 that in S.
cerevisiae homologous chromosomes pair dependent on the interaction switch from
non-homologous centromeres to homologous centromeres. In S. cerevisiae, Zip1 and
Spo11 are two proteins proved to be required for centromeric interactions and the
transition from non-homologous to homologous centromeres pairing respectively
(Tsubouchi et al., 2005). Nsl1p is another protein with a role in transient centromeres
pairing in S.cerevisiae (Nekrasov et al., 2002). However in Drosophila, the
mechanism of homologous centromeres pairing is not clear and no proteins have been
found.
In our research, we first found that homologous conjunction protein SNM, but
not MNM, is involved in homologous centromeres pairing in the stage S3 in PI. In
snm mutations homologous centromeres are unpaired and two CID spots can be seen
in each territory, compared to wild-type, in which centromeres transiently pair from
S3 to S4 stage and each territory contains one spot, indicating that centromere pairing
is disrupted at the absence of SNM. As a homolog of SCC3 / REC11 and SA/STAG
proteins, which are components of cohesin (Prieto et al., 2001; Kitajima et al., 2003),
SNM was previously found to have no role in sister chromatid cohesion (Thomas et
73
al., 2005), however it still provides a rule for us to explore the relationship between
cohesin complex and homologous centromeres pairing. In addition, Kemp et al. in
2004 provided evidence that in budding yeast centromeres pairing is required for an
achiasmate segregation in meiosis (Kemp et al., 2004). Here we show that SNM is
required for centromeres pairing, which suggests there may be a conserved pathway
in achiasmate segregation.
Mutations in sun and snm interact to cause sister chromatid separation in
mid-prophase (S3), which is earlier than single sun mutants. Taken together with the
different morphology of chromosomes in single sun mutants and sun and snm double
mutants, we suggest SNM has another function in addition to homolog conjunction
and plays a role in sister centromere cohesion and in meiotic centromere cohesion.
2. How cohesion and pairing proteins affect the integrity of chromosome
territories?
Chromosome territories correspond to confined chromosomes in discrete
regions within nuclei. Their distinct properties have been extensively discussed and
are important for gene regulation and genome stability in health and disease (Meaburn
et al., 2007). However, little is known about the forces that determine how
chromosomes are organized. In Drosophila three large bivalents are organized into
three separated territories throughout PI. Vazquez et al., proposed in 2002 that the
territories are defined by connections between the chromosomes and components of
the nuclear envelope (Vazquez et al., 2002). However from our research, we found
74
that territories retained normal morphology in singule sun mutants, but exhibited
abnormally diffuse in mnm and snm mutants throughout PI, uncovering that that SNM
and MNM are involved in normal territory formation and restriction during PI. In
addition, loss of both SUN and MNM or both SUN and SNM impair severely the
integrity of chromosome territories, which implied that sister chromatid cohesion and
homologous conjunction contribute to territory structures. How these two systems
work together to affect normal chromosome territories formation is enigma. Our
explanation here is loss of both sister chromatid cohesion and homologous
conjunction proteins completely separate the sister chromatids, resulting in a complete
subdivision of territories.
75
Chapter V - References
76
Ahmad K., Engel C.K., Prive G.G. (1998). Crystal structure of the BTB domain from
PLZF. Proc Natl Acad Sci U S A. Oct 13;95(21):12123-8.
Ahmad K., Henikoff S. (2001). Centromeres are specialized replication domains in
heterochromatin. J. Cell Biol. 153, 101-110.
Ahmad K., Melnick A., Lax S., Bouchard D., Liu J., Kiang C.L., Mayer S., Takahashi
S., Licht J.D., Prive G.G. (2003). Mechanism of SMRT corepressor recruitment by the
BCL6 BTB domain. Mol Cell. Dec;12(6):1551-64.
Albagli O., Dhordain P., Deweindt C., Lecocq G., Leprince D. (1995). The BTB/POZ
domain: a new protein-protein interaction motif common to DNA- and actin-binding
proteins. Cell Growth Differ. Sep;6(9):1193-8.
Allers T., Lichten M. (2001). Differential timing and control of noncrossover and
crossover recombination during meiosis. Cell. Jul 13;106(1):47-57.
Anderson L.K., Royer S.M., Page S.L., McKim K.S., Lai A., Lilly M.A., Hawley R.S.
(2005). Juxtaposition of C(2)M and the transverse filament protein C(3)G within the
central region of Drosophila synaptonemal complex. Proc Natl Acad Sci U S A Mar
22;102(12):4482-7
77
Arumugam, P., Gruber, S., Tanaka, K., Haering, C. H., Mechtler, K., Nasmyth, K.
(2003). ATP hydrolysis is required for cohesin’s association with chromosomes. Curr.
Biol. 13, 1941–1953.
Bai C., Sen P., Hofmann K., Ma L., Goebl M., Harper J.W., Elledge S.J. (1996). SKP1
connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel
motif, the F-box. Cell. Jul 26;86(2):263-74.
Balicky E.M., Endres M.W., Lai C., Bickel S.E. (2002). Meiotic cohesion requires
accumulation of ORD on chromosomes before condensation. Mol. Biol. Cell 21,
3890–3900.
Beaster-Jones L., Okkema P.G. (2004). DNA binding and in vivo function of
C.elegans PEB-1 require a conserved FLYWCH motif. J Mol Biol. Jun
11;339(4):695-706.
Bergerat A., Massy B., Gadelle D., Varoutas P. C., Nicolas A., Forterre P. (1997). An
atypical topoisomerase II from archaea with implications for meiotic recombination.
Nature 386, 414 – 417.
Bickel S.E., Orr-Weaver T.L., Balicky E.M. (2002). The sister-chromatid cohesion
protein ORD is required for chiasma maintenance in Drosophila oocytes. Curr Biol.
78
Jun 4;12(11):925-9.
Bickel S.E., Wyman D.W., Miyazaki W.Y., Moore D.P., Orr-Weaver T.L. (1996).
Identification of ORD, a Drosophila protein essential for sister chromatid cohesion.
EMBO J. Mar 15;15(6):1451-9.
Bickel S.E., Wyman D.W., Orr-Weaver T.L. (1997). Mutational analysis of the
Drosophila sister-chromatid cohesion protein ORD and its role in the maintenance of
centromeric cohesion. Genetics. Aug;146(4):1319-31
Blower M.D., Karpen G.H. (2001). The role of Drosophila CID in kinetochore
formation, cell-cycle progression and heterochromatin interactions. Nat. Cell Biol. 3,
730-739.
Bonaccorsi, S., Giansanti, M.G., Cenci, G., Gatti, M. (2000). Cytological analysis of
spermatocyte growth and male meiosis in Drosophila melanogaster. In Drosophila
Protocols, M. Ashburner, R. Scott Hawley, and W. Sullivan, eds. (New York: Cold
Spring Harbor Press).
Botuyan M.V., Mer G., Yi G.S., Koth C.M., Case D.A., Edwards A.M., Chazin W.J.,
Arrowsmith C.H. (2001). Solution structure and dynamics of yeast elongin C in
complex with a von Hippel-Lindau peptide. J Mol Biol. Sep 7;312(1):177-86.
79
Brian L., Amon A. (2001). Meiosis: How to create a specialized cell cycle. Curr Opin
Cell Biol. Dec;13(6):770-7.
Buchner K., Roth P., Schotta G., Krauss V., Saumweber H., Reuter G., Dorn R. (2000).
Genetic and molecular complexity of the position effect variegation modifier
mod(mdg4) in Drosophila. Genetics 155, 141–157.
Buonomo S.B.C., Clyne R.K., Fuchs J., Loidl J., Uhlmann F., Nasmyth K. (2000).
Disjunction of homologous chromosomes in meiosis I depends on proteolytic
cleavage of the meiotic cohesin Rec8 by separin. Cell; 103:387-98.
Cenci, G., Bonaccorsi, S., Pisano, C., Verni, F., Gatti, M. (1994). Chromatin and
microtubule organization during premeiotic, meiotic and early postmeiotic stages of
Drosophila melanogaster spermatogenesis. J. Cell Sci. 107, 3521–3534.
Ciosk R., Shirayama M., Shevchenko A., Tanaka T., Toth A., Shevchenko A.,
Nasmyth K. (2000). Cohesin's binding to chromosomes depends on a separate
complex consisting of Scc2 and Scc4 proteins. Mol Cell. Feb;5(2):243-54.
Ciosk, R., Zachariae W., Michaelis C., Shevchenko A., Mann M., Nasmyth K. (1998).
An Esp1/Pds1 complex regulates loss of sister chromatid cohesion at the metaphase to
anaphase transition in yeast. Cell. 93:1067–1076
80
Clarke A.S., Tang T.T., Ooi D.L., Orr-Weaver T.L. (2005). POLO kinase regulates the
Drosophila centromere cohesion protein MEI-S332. Dev Cell. Jan;8(1):53-64.
Cohen-Fix, O., Peters J.M., Kirschner M.W., Koshland D. (1996). Anaphase initiation
in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the
anaphase inhibitor Pds1p. Genes Dev. 10:3081–3093
Cohen P., Pollard J.W. (2001). Regulation of meiotic recombination and prophase I
progression in mammals. BioEssays 23:996-1009.
Cooper K.W. (1964). Meiotic conjunctive elements not involving chiasmata. Proc
Natl Acad Sci U S A. Nov;52:1248-55.
Davis, B. (1971). Genetic analysis of a meiotic mutant resulting in precocious
sister-centromere separation in Drosophila melanogaster. Mol. Gen. Genet. 113,
251-272.
Dorn R., Krauss V. (2003). The modifier of mdg4 locus in Drosophila: functional
complexity is resolved by trans splicing. Genetica. Mar;117(2-3):165-77.
Dorn R., Krauss V., Reuter G., Saumweber, H. (1993). The enhancer of position-effect
variegation of Drosophila, E(var)3–93D, codes for a chromatin protein containing a
81
conserved domain common to several transcriptional regulators. Proc. Natl. Acad. Sci.
USA 90, 11376–11380.
Funabiki, H., Yamano H., Kumada K., Nagao K., Hunt T., anagida Y.M. (1996b).
Cut2 proteolysis required for sister-chromatid separation in fission yeast. Nature.
381:438–441.
Furukawa M., He Y.J., Borchers C., Xiong Y. (2003). Targeting of protein
ubiquitination by BTB-Cullin 3-Roc1 ubiquitin ligases. Nat Cell Biol;5:1001–1007.
Gause M., Morcillo P., Dorsett D. (2001). Insulation of enhancer–promoter
communication by a gypsy transposon insert in the Drosophila cut gene: cooperation
between suppressor of hairywing and modifier of mdg4 proteins. Mol. Cell. Biol.
21:4807–4817.
Gerasimova A. (1995). A Drosophila protein that imparts directionality on a
chromatin insulator is an enhancer of position-effect variegation. Cell 82, 587–597.
Ghabrial A., Ray R.P., Schupbach T. (1998). okra and spindle-B encode components
of the RAD52 DNA repair pathway and affect meiosis and patterning in Drosophila
oogenesis. Genes Dev. Sep 1;12(17):2711-23.
82
Ghosh, D., Gerasimova T.I., Corces V.G. (2001). Interactions between the Su(Hw) and
Mod(mdg4) proteins required for gypsy insulator function. EMBO J. 20: 2518–2527.
Gimenez-Abian J.F., Sumara I., Hirota T., Hauf S., Gerlich D., de la Torre C.,
Ellenberg J., Peters J.M. (2004). Regulation of sister chromatid cohesion between
chromosome arms. Curr Biol. Jul 13;14(13):1187-93.
Glynn E.F., Megee P.C., Yu H.G., Mistrot C., Unal E., Koshland D.E., DeRisi J.L.,
Gerton J.L. (2004). Genome-wide mapping of the cohesin complex in the yeast
Saccharomyces cerevisiae. PLoS Biol. Sep;2(9):E259. Epub Jul 27.
Godt D., Couderc J.L., Cramton S.E., Laski F.A. (1993). Pattern formation in the
limbs of Drosophila: bric a brac is expressed in both a gradient and a wave-like
pattern and is required for specification and proper segmentation of the tarsus.
Development 119:799–812.
Goldstein, L.S.B. (1980). Mechanisms of chromosome orientation revealed by two
meiotic mutants in Drosophila melanogaster. Chromosoma 78, 79-111.
Gorczyca M., Popova E., Jia X.X., Budnik V. (1999). The gene mod(mdg4) affects
synapse specificity and structure in Drosophila. J. Neurobiol. 39, 447–460.
83
Gruber S., Haering C.H., Nasmyth K. (2003). Chromosomal cohesin forms a ring.
Cell. Mar 21;112(6):765-77.
Gruber S., Arumugam P., Katou Y., Kuglitsch D., Helmhart W., Shirahige K.,
Nasmyth K. (2006). Evidence that loading of cohesin onto chromosomes involves
opening of its SMC hinge. Cell. Nov 3;127(3):523-37.
Haering, C.H. (2002). Molecular architecture of SMA proteins and yeast cohein
complex. Mol. Cell 9, 773-788.
Hamant O., Golubovskaya I., Meeley R., Fiume E., Timofejeva L., Schleiffer A.,
Nasmyth K., Cande W.Z. (2005). A REC8-dependent plant Shugoshin is required for
maintenance of centromeric cohesion during meiosis and has no mitotic functions.
Curr. Biol. 15:948–954
Harvey A.J., Bidwai A.P., Miller L. K. (1997). Doom, a product of the Drosophila
mod(mdg4) gene, induces apoptosis and binds to baculovirus inhibitor-of-apoptosis
proteins. Mol. Cell. Biol. 17:2835–2843
Hassold T., Hunt P. (2001). To err (meiotically) is human: the genesis of human
aneuploidy. Nat. Rev. Genet. 2, 280–291.
84
Hauf, S., Roitinger, E., Koch, B., Dittrich, C. M., Mechtler, K. Peters, J.-M. (2005).
Dissociation of cohesin from chromosome arms and loss of arm cohesion during early
mitosis depends on phosphorylation of SA2. PLOS Biol. 3, e69
Hauf S., Watanabe Y. (2004). Kinetochore orientation in mitosis and meiosis.
Cell. Oct 29;119(3):317-27. Review.
Heidmann D., Horn S., Heidmann S., Schleiffer A., Nasmyth K., Lehner C.F. (2004).
The Drosophila meiotic kleisin C(2)M functions before the meiotic divisions.
Chromosoma. Oct;113(4):177-87.
Hirano M., Anderson D.E., Erickson H.P., Hirano T. (2001). Bimodal activation of
SMC ATPase by intra- and inter-molecular interactions. EMBO J. 20 pp. 3238–3250
Hirano T. (2006). At the heart of the chromosome: SMC proteins in action Nat Rev
Mol Cell Biol. May;7(5):311-22. Review
Hunter N., Kleckner N. (2001). The single-end invasion: an asymmetric intermediate
at the double-strand break to double-holliday junction transition of meiotic
recombination. Cell. Jul 13; 106(1):59-70.
Indjeian V.B., Stern B.M., Murray A.W. (2005). The centromeric protein Sgo1 is
85
required to sense lack of tension on mitotic chromosomes. Science. Jan
7;307(5706):130-3.
Ishiguro K., Watanabe Y. (2007). Chromosome cohesion in mitosis and meiosis. J Cell
Sci. Feb 1;120(Pt 3):367-9. Review.
James P., Halladay J., Craig E.A. (1996).Genomic libraries and a host strain designed
for highly efficient two-hybrid selection in yeast. Genetics. Dec;144(4):1425-36.
Kang M.I., Kobayashi A., Wakabayashi N., Kim S.G., Yamamoto M. (2004).
Scaffolding of Keap1 to the actin cytoskeleton controls the function of Nrf2 as key
regulator of cytoprotective phase 2 genes. Proc Natl Acad Sci USA. 101:2046–2051.
Karpen G.H., Le M.H., Le H. (1996). Centric heterochromatin and the efficiency of
achiasmate
disjunction
in
Drosophila
female
meiosis.
Science.
Jul;5
273(5271):118-22
Katis V.L., Galova M., Rabitsch K.P., Gregan J., Nasmyth K. (2004). Maintenance of
cohesin
at
centromeres
after
meiosis
I
in
budding
yeast
requires
a
kinetochore-associated protein related to MEI-S332. Curr. Biol. 14, 560-572.
Keeney S. (2001). Mechanism and control of meiotic recombination initiation. Curr
86
Top Dev Biol. 52:1-53.
Keeney S., Giroux C.N., Kleckner N. (1997). Meiosis-specific DNA double-strand
breaks are catalyzed by Spo11, a member of a widely conserved protein family. Cell.
Feb 7; 88 (3):375-84.
Kemp B., Boumil R.M., Stewart M.N., Dawson D.S. (2004). A role for centromere
pairing in meiotic chromosome segregation. Genes Dev. Aug 15;18(16):1946-51.
Kerrebrock A.W., Miyazaki W.Y., Birnby D., Orr-Weaver T.L. (1992). The Drosophila
mei-S332 gene promotes sister-chromatid cohesion in meiosis following kinetochore
differentiation. Genetics 130, 827-841.
Kerrebrock A.W., Moore D.P., Wu J.S., Orr-Weaver T.L. (1995). Mei-S332, a
Drosophila protein required for sister-chromatid cohesion, can localize to meiotic
centromere regions. Cell. Oct 20;83(2):247-56.
Kiburz B.M., Reynolds D.B., Megee P.C., Marston A.L., Lee B.H., Lee T.I., Levine
S.S., Young R.A., Amon A. (2005). The core centromere and Sgo1 establish a 50-kb
cohesin-protected domain around centromeres during meiosis I. Genes Dev.
19:3017–3030.
87
Kim T.A., Jiang S., Seng S., Cha K., Avraham H.K., Avraham S. (2005). The BTB
domain of the nuclear matrix protein NRP/B is required for neurite outgrowth. J Cell
Sci. Dec 1;118(Pt 23):5537-48. Erratum in: J Cell Sci. Jan 1;119(Pt 1):195.
Kitajima T.S., Hauf S., Ohsugi M., Yamamoto T., Watanabe Y. (2005). Human Bub1
defines the persistent cohesion site along the mitotic chromosome by affecting
shugoshin localization. Curr Biol 15: 353-359.
Kitajima T.S., Kawashima S.A., Watanabe Y. (2004). The conserved kinetochore
protein shugoshin protects centromeric cohesion during meiosis. Nature 427, 510-517.
Kitajima T. S., Sakuno T., Ishiguro K., Iemura S., Natsume T., Kawashima S. A.,
Watanabe Y. (2006). Shugoshin collaborates with protein phosphatase 2A to protect
cohesin. Nature 441, 46-52.
Kitajima T.S., Yokobayashi S., Yamamoto M., Watanabe Y. (2003). Distinct cohesin
complexes organize meiotic chromosome domains. Science 300, 1152–1155.
Kleckner N. (1996). Meiosis: how could it work? Proc Natl Acad Sci U S A. Aug.
6;93(16):8167-74.
Klein F., Mahr P., Galova M., Buonomo S.B., Michaelis C., Nairz K., Nasmyth K.
88
(1999). A central role for cohesins in sister chromatid cohesion, formation of axial
elements, and recombination during yeast meiosis. Cell. Jul 9;98(1):91-103.
Knowles B.A., Hawley R.S. (1991). Genetic analysis of microtubule motor proteins in
Drosophila: a mutation at the ncd locus is a dominant enhancer of nod. Proc Natl
Acad Sci U S A. Aug 15;88(16):7165-9.
Koonin E.V., Senkevich T.G., Chernos V.I. (1992). A family of DNA virus genes that
consists of fused portions of unrelated cellular genes. Trends Biochem Sci
17:213–214.
Koundakjian E.J., Cowan D.M., Hardy R.W., Becker A.H. (2004). The Zuker
collection: a resource for the analysis of autosomal gene function in Drosophila
melanogaster. Genetics 167, 203–206.
Kreusch A., Pfaffinger P.J., Stevens C.F., Choe S. (1998). Crystal structure of the
tetramerization domain of the Shaker potassium channel. Nature.;392:945–948.
Lake C.M., Hawley R.S. (2005). A new target for POLO in meiotic centromere
cohesion. Dev Cell. Jan;8(1):5-7.
Lam W.W., Peterson E.A., Yeung M., Lavoie B.D. (2006). Condensin is required for
89
chromosome arm cohesion during mitosis. Genes Dev. Nov 1;20(21):2973-84.
Lee B.H., Amon A., Prinz S. (2002). Spo13 regulates cohesin cleavage. Genes Dev.
16, 1672-1681.
Lee J.Y., Dej K.J., Lopez J.M., Orr-Weaver T.L. (2004). Control of centromere
localization of the MEI-S332 cohesion protection protein. Curr. Biol. 14, 1277-1283.
Lee J.Y., Orr-Weaver T.L. (2001). The molecular basis of sister-chromatid cohesion.
Annu Rev Cell Dev Biol.;17:753-77.
Lee J.Y., Hayashi-Hagihara A., Orr-Weaver T.L. (2005). Roles and regulation of the
Drosophila centromere cohesion protein MEI-S332 family. Philos Trans R Soc Lond
B Biol Sci. Mar 29;360(1455):543-52.
Lengronne A., Katou Y., Mori S., Yokobayashi S., Kelly G.P., Itoh T., Watanabe Y.,
Shirahige K., Uhlmann F. (2004). Cohesin relocation from sites of chromosomal
loading to places of convergent transcription. Nature. Jul 29;430(6999):573-8.
Losada A., Hirano T. (2005). Dynamic molecular linkers of the genome: the first
decade of SMC proteins. Genes Dev. 19, 1269-1287.
Mackay J.P., Crossley M. (1998). Zinc fingers are sticking together. Trends Biochem
90
Sci. Jan;23(1):1-4.
Manheim E.A., McKim K.S. (2003). The Synaptonemal complex component C(2)M
regulates meiotic crossing over in Drosophila. Curr Biol. Feb 18;13(4):276-85.
Mazur A.M., Georgiev P.G., Golovnin A.K. (2005). Analysis of interaction between
proteins containing the BTB domain in the yeast two-hybrid system. Dokl Biochem
Biophys. Jan-Feb;400:24-7.
Meaburn K.J., Misteli T. (2007). Cell biology: chromosome territories. Nature 445:
379-381.
McGuinness B., Hirota T., Kudo N.R., Peters J.M., Nasmyth K. (2005). Shugoshin
prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells.
PLoS Biol 3: e86
McKee B.D. (2004). Homologous pairing and chromosome dynamics in meiosis and
mitosis. Biochim Biophys Acta. Mar 15;1677(1-3):165-80.
McKee B.D., Habera L., Vrana J.A. (1992). Evidence that intergenic spacer repeats of
Drosophila melanogaster rRNA genes function as X-Y pairing sites in male meiosis,
and a general model for achiasmatic pairing. Genetics. Oct;132(2):529-44.
91
McKee B.D., Lumsden S.E., Das S. (1993). The distribution of male meiotic pairing
sites on chromosome 2 of Drosophila melanogaster: Meiotic pairing and segregation
of 2-Y transpositions. Chromosoma 102: 180-194.
McKim K.S., Jang J.K., Manheim E.A. (2002). Meiotic recombination and
chromosome segregation in Drosophila females. Annu Rev Genet.36:205-32.
Melnick A., Ahmad K.F., Arai S., Polinger A., Ball H., Borden K.L., Carlile G.W.,
Prive G.G., Licht J.D. (2000). In-depth mutational analysis of the promyelocytic
leukemia zinc finger BTB/POZ domain reveals motifs and residues required for
biological and transcriptional functions. Mol Cell Biol. Sep;20 (17):6550-67.
Milutinovich M., Unal E., Ward C., Skibbens R.V., Koshland D. (2007). A Multi-Step
Pathway for the Establishment of Sister Chromatid Cohesion.PLoS Genet. Jan
19;3(1):e12
Minor D.L., Lin Y.F., Mobley B.C., Avelar A., Jan Y.N., Jan L.Y., Berger J.M. (2000).
The polar T1 interface is linked to conformational changes that open the voltage-gated
potassium channel. Cell.102:657–670.
Miyazaki W.Y., Orr-Weaver T. L. (1992). Sister-chromatid misbehavior in Drosophila
ord mutants. Genetics. Dec;132 (4):1047-61.
92
Miyazaki W. Y., Orr-Weaver T. L. (1994). Sister chromatid cohesion in mitosis and
meiosis. Annual Review of Genetics v28. pp167(21).
Moore D.P., Page A.W., Tang T.T., Kerrebrock A.W., Orr-Weaver T.L. (1998). The
cohesion protein MEI-S332 localizes to condensed meiotic and mitotic centromeres
until sister chromatids separate. J Cell Biol. Mar 9;140(5):1003-12
Morris J., Lehmann R. (1999). Drosophila oogenesis: versatile spn doctors. Curr Biol.
Jan 28;9(2):R55-8.
Neale M.J., Pan J., Keeney S. (2005). Endonucleolytic processing of covalent
protein-linked DNA double-strand breaks. Nature 436:1053–1057.
Nekrasov V.S., Smith M.A., Peak-Chew S., Kilmartin J.V. (2003). Interactions
between centromere complexes in Saccharomyces cerevisiae. Mol Biol Cell.
Dec;14(12):4931-46
Orr-Weaver T.L. (1995). Meiosis in Drosophila: seeing is believing. Proc Natl Acad
Sci U S A. Nov 7;92(23):10443-9.
Perez-Torrado R., Yamada D., Defossez P.A. (2006). Born to bind: the BTB
protein-protein interaction domain. Bioessays. Dec;28(12):1194-202.
93
Petronczki M., Siomos M.F., Nasmyth K. (2003). Un menage a quatre: the molecular
biology of chromosome segregation in meiosis.Cell. Feb 21;112(4):423-40. Review.
Pidoux A.L., Allshire R.C. (2004). Kinetochore and heterochromatin domains of the
fission yeast centromere. Chromosome Res.12(6):521-34. Review.
Pintard L., Willis J.H., Willems A., Johnson J.L., Srayko M., Kurz T., Glaser S.,
Mains P.E., Tyers M., Bowerman B., Peter M. (2003). The BTB protein MEL-26 is a
substrate-specific adaptor of the CUL-3 ubiquitin-ligase. Nature.425:311–316.
Prieto I., Suja J.A., Pezzi N., Kremer L., Martinez A.C., Rufas J.S., Barbero J.L.
(2001). Mammalian STAG3 is a cohesin specific to sister chromatid arms in meiosis I.
Nat. Cell Biol. 3, 761–766.
Resnick T.D., Satinover D.L., MacIsaac F., Stukenberg P.T., Earnshaw W.C.,
Orr-Weaver T.L. (2006). Carmena M.INCENP and Aurora B promote meiotic sister
chromatid cohesion through localization of the Shugoshin MEI-S332 in Drosophila.
Dev Cell. Jul;11(1):57-68
Revenkova E., Eijpe M., Heyting C., Hodges C.A., Hunt P.A., Liebe B., Scherthan H.,
Jessberger R. (2004). Cohesin SMC1 beta is required for meiotic chromosome
dynamics, sister chromatid cohesion and DNA recombination. Nat Cell Biol.
94
Jun;6(6):555-62.
Riedel C. G., Katis V. L., Katou Y., Mori S., Itoh T., Helmhart W., Galova M.,
Petronczki M., Gregan J., Cetin B. (2006). Protein phosphatase 2A protects
centromeric sister chromatid cohesion during meiosis I. Nature 441, 53-61
Schleiffer A. (2003). Kleisins: a superfamily of bacterial and eukaryotic SMC protein
partners, Mol. Cell 11 pp. 571–575
Sheu
D.S.,
Wan
Y.T.,
Lee.
C.Y.
(2000).
Rapid
detection
of
polyhydroxyalkanoate-accumulating bacteria isolated from the environment by
colony PCR. Microbiology 146, 2019-2025.
Shonn M.A., McCarroll R., Murray A.W. (2002). Spo13 protects meiotic cohesin at
centromeres in meiosis I. Genes Dev. 16, 1659-1671.
Sjogren C., Nasmyth K. (2001). Sister chromatid cohesion is required for
postreplicative double-strand break repair in Saccharomyces cerevisiae, Curr. Biol. 11,
pp. 991–995.
Skibbens R.V., Corson L.B., Koshland D., Hieter P. (1999). Ctf7p is essential for
sister chromatid cohesion and links mitotic chromosome structure to the DNA
95
replication machinery. Genes Dev. Feb 1;13(3):307-19.
Soltani-Bejnood M., Thomas S.E., Villeneuve L., Schwartz K., Hong C.S.,
McKee B.D. (2007). Role of the mod(mdg4) Common Region in Homolog
Segregation in Drosophila Male Meiosis. Genetics. May;176(1):161-80
Stemmann, O., Zou H., Gerber S.A., Gygi S.P., Kirschner M.W. (2001). Dual
inhibition of sister chromatid separation at metaphase. Cell. 107:715–726.
Stogios P.J., Downs G.S., Jauhal J.J., Nandra S.K., Prive G.G. (2005). Sequence and
structural analysis of BTB domain proteins. Genome Biol. 6(10):R82.
Stoop-Myer C., Amon A. (1999). Meiosis: Rec8 is the reason for cohesion.
Nat Cell Biol. Sep;1(5):E125-7
Sumara I., Vorlaufer E., Stukenberg P.T., Kelm O., Redemann N., Nigg E.A., Peters
J.M. (2002). The dissociation of cohesin from chromosomes in prophase is regulated
by Polo-like kinase. Mol Cell. Mar;9(3):515-25.
Tang T.T., Bickel S..E., Young L.M., Orr-Weaver T.L. (1998). Maintenance of
sister-chromatid cohesion at the centromere by the Drosophila MEI-S332 protein.
Genes Dev. 1998 Dec 15;12(24):3843-56.
96
Tang Z., Sun Y., Harley E., Zou H., Yu H. (2004). Human Bub1 protects centromeric
sister-chromatid cohesion through Shugoshin during mitosis. Proc Natl Acad Sci USA
101: 1812-1817
Tang Z., Shu H., Qi W., Mahmood N. A., Mumby M. C. Yu H. (2006). PP2A is
required for centromeric localization of Sgo1 and proper chromosome segregation.
Dev. Cell 10, 575-585.
Thomas S.E., Soltani-Bejnood M., Roth P., Dorn R., Logsdon J.M. Jr., McKee B.D.
(2005). Identification of two proteins required for conjunction and regular segregation
of achiasmate homologs in Drosophila male meiosis. Cell. Nov 18;123(4):555-68.
Tomkiel J.E., Wakimoto B.T., Briscoe, A. Jr. (2001). The Teflon gene is required for
maintenance of autosomal homolog pairing at meiosis I in male Drosophila
melanogaster. Genetics 157, 273–281.
Toth A., Ciosk R., Uhlmann F., Galova M., Schleiffer A., Nasmyth K. (1999). Yeast
cohesin complex requires a conserved protein, Eco1p(Ctf7), to establish cohesion
between sister chromatids during DNA replication. Genes Dev. Feb 1;13(3):320-33.
Toth, A., Rabitsch, K.P., Galova, M., Schleiffer, A., Buonomo, S.B., Nasmyth, K.
(2000). Functional genomics identifies monopolin: akinetochore protein required for
97
segregation of homologs during meiosis I. Cell 103, 1155-1168.
Tsubouchi T., Roeder G.S. (2005). synaptonemal complex protein promotes
homology-independent centromere coupling. Science May 6;308(5723):870-3.
Uhlmann F., Lottspeich F., Nasmyth K. (1999). Sister chromatid separation at
anaphase onset is promoted by cleavage of the cohesin subunit Scc1p. Nature.
400:37–42
Vazquez J., Belmont A. S., Sedat J. W. (2002). The Dynamics of 68. Homologous
Chromosome Pairing during Male Drosophila Meiosis. Current Biol 12: 1473-1483.
Waizenegger I., Hauf S., Meinke A., Peters J.M. (2000). Two distinct pathways
remove mammalian cohesin from chromosome arms in prophase and from
centromeres in anaphase. Cell.103:399–410.
Walker M.Y., Hawley R.S. (2000). Hanging on to your homolog: the roles of pairing,
synapsis and recombination in the maintenance of homolog adhesion. Chromosoma:
109(1-2):3-9.
Wakimoto B.T., Lindsley D.L., Herrera C. (2004). Toward a comprehensive genetic
analysis of male fertility in Drosophila melanogaster. Genetics 167: 207-216.
98
Wang Z., Castano I.B., De Las Penas A., Adams C., Christman M.F. (2000). Pol
kappa: A DNA polymerase required for sister chromatid cohesion. Science. Aug
4;289(5480):774-9.
Watanabe Y. (2005). Shugoshin: guardian spirit at the centromere. Curr. Opin. Cell
Biol. 17, 590-595
Watanabe Y, Nurse P. (1999). Cohesin Rec8 is required for reductional chromosome
segregation at meiosis. Nature. Jul 29;400(6743):461-4.
Webber H.A., Howard L., Bickel S.E. (2004). The cohesion protein ORD is required
for homologue bias during meiotic recombination. J Cell Biol. Mar 15;164(6):819-29.
Weitzer S., Lehan C., Uhlmann, F. (2003). A model for ATP hydrolysis-dependent
binding of cohesin to DNA. Curr. Biol. 13, 1930–1940.
Wilkins A., Ping Q., Carpenter C.L. (2004). RhoBTB2 is a substrate of the
mammalian Cul3 ubiquitin ligase complex. Genes Dev.18:856–861.
Xu H., Beasley M.D., Warren W.D., van der Horst G.T., McKay M.J. (2005). Absence
of mouse REC8 cohesin promotes synapsis of sister chromatids in meiosis. Dev Cell.
Jun;8(6):949-61.
99
Yamamoto A., Hiraoka Y. (2003). Monopolar spindle attachment of sister chromatids
is ensured by two distinct mechanisms at the first meiotic division in fission
yeast.EMBO J. May 1;22(9):2284-96.
Yokobayashi S., Watanabe Y. (2005). The kinetochore protein Moa1 enables
cohesion-mediated monopolar attachment at meiosis I. Cell 123, 803-817.
Yokobayashi S., Yamamoto M., Watanabe Y. (2003). Cohesins determine the
attachment manner of kinetochores to spindle microtubules at meiosis I in fission
yeast. Mol Cell Biol. Jun;23(11):3965-73.
Yoo S., McKee B.D. (2004). Overexpression of Drosophila Rad51 protein (DmRad51)
disrupts
cell
cycle
progression
and
leads
to
apoptosis.
Chromosoma.
Sep;113(2):92-101.
Yu H.G., Koshland D. (2005). Chromosome morphogenesis: condensin-dependent
cohesin removal during meiosis. Cell. Nov 4;123(3):397-407.
Zickler D., Kleckner N. (1999). Meiotic chromosomes: integrating structure and
function. Annu Rev Genet. 33:603-754.
Ziegelbauer J., Shan B., Yager D., Larabell C., Hoffmann B., Tjian R. (2001).
100
Transcription factor MIZ-1 is regulated via microtubule association. Mol Cell.
8:339–349.
Zollman S., Godt D., Prive G.G., Couderc J.L., Laski F.A. (1994). The BTB domain,
found primarily in zinc finger proteins, defines an evolutionarily conserved family
that includes several developmentally regulated genes in Drosophila. Proc Natl Acad
Sci. USA 91:10717–10721.
Zou H., McGarryT.J., Bernal T., Kirschner M.W. (1999). Identification of a vertebrate
sister-chromatid separation inhibitor involved in transformation and tumorigenesis.
Science. 285:418–422.
101
Vita
Jian Ma was born in March 1975 in Wujin, China. After graduated from Qian-Huang
high school, he attended NanJing Medical University in 1994 and earned his M.D.
degree in 1999. From 1999 to 2001 he worked in NanJing Brain Hospital as a
psychiatrist, and after that, he studied in the Shanghai Reproductive Medicine Key
Laboratory as a research assistant until 2004. Jian began his master studies in the fall
of 2004 at the University of Tennessee, Knoxville. He defended his thesis in July
2007.
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