BA_finalohneMovies

BACHELOR THESIS
Methylation dynamics
during Meiosis in
Arabidopsis thaliana (L.)
Frederike Schäfer
Bachelor of Science Biology
University of Hamburg
handed in: August 2017
1. Corrector: Prof. Dr. Arp Schnittger
2. Corrector: Kostika Sofroni

Contents
1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2 Zusammenfassung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.1 General function of DNA methylation . . . . . . . . . . . . . . . . . . 7
3.2 Establishment, maintenance and function of DNA methylation . . . . . 8
3.3 Methylation during Meiosis . . . . . . . . . . . . . . . . . . . . . . . 13
3.4 Visualization of Methylation - DYNAMET reporters . . . . . . . . . . 15
3.5 Aim of this project . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
4 Material and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
4.1 Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
4.2 Molecular methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
4.3 Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
4.4 Confocal microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
5 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
5.1 Detection of CG methylation with MBD6 −GFP . . . . . . . . . . . . . 28
5.2 Detection of CHH methylation with SUVH9 −GFP . . . . . . . . . . . 38
6 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
6.1 Comparison of MBD6 −GFP and SUVH9 −GFP dynamics . . . . . . . . 42
6.2 Dynamics of MBD6 −GFP . . . . . . . . . . . . . . . . . . . . . . . . 43
6.3 Dynamics of SUVH9 −GFP . . . . . . . . . . . . . . . . . . . . . . . . 46
6.4 General remarks on the use of fluorescent proteins . . . . . . . . . . . 48
6.5 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
6.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
7 Eidesstaatliche Erklärung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
8 Supplementaries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.1 Movies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.2 Additional graphics . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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8.3 Duration of meiotic substages . . . . . . . . . . . . . . . . . . . . . .
8.4 Vector Sequences and Maps . . . . . . . . . . . . . . . . . . . . . . .
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List of Figures
1 RNA-directed DNA methylation pathway . . . . . . . . . . . . . . . . . . . . 9
2 Structures of CMT3 and CMT2 . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3 mCG-Venus during male sporogenesis . . . . . . . . . . . . . . . . . . . . . . 16
4 mCHH-Venus during male sporogenesis . . . . . . . . . . . . . . . . . . . . . 17
5 Multisite Gateway Three-Fragment Vector construction . . . . . . . . . . . . . 23
6 Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
7 MBD6 −GFP during pre-meiosis . . . . . . . . . . . . . . . . . . . . . . . . . 29
8 MBD6 −GFP xREC8 −mRUBY 3 during pre-meiosis . . . . . . . . . . . . . . . . . 29
9 MBD6 −GFP x TagRFP− CENH3 in DE +/− during pre-meiosis . . . . . . . . . . . 30
10 MBD6 −GFP during early prophase . . . . . . . . . . . . . . . . . . . . . . . . 31
11 Colocalization of MBD6 −GFP with REC8 −mRUBY 3 . . . . . . . . . . . . . . . 32
12 MBD6 −GFP during early to mid-prophase . . . . . . . . . . . . . . . . . . . . 33
13 MBD6 −GFP xREC8 −mRUBY 3 during mid-prophase . . . . . . . . . . . . . . . . 33
14 MBD6 −GFP xASY3 −TagRFP during Mid-prophase . . . . . . . . . . . . . . . . 34
15 MBD6 −GFP x ASY3 −TagRFP during middle to end of prophase . . . . . . . . . 35
16 MBD6 −GFP xASY3 −TagRFP during Prophase I to Metaphase I transition . . . . 35
17 MBD6 −GFP during anaphase I to telophase I transition . . . . . . . . . . . . . 36
18 MBD6 −GFP during prophase II to metaphase II transition . . . . . . . . . . . . 36
19 MBD6 −GFP during metaphase II to telophase II transition . . . . . . . . . . . . 37
20 SUVH9 −GFP during pre-meiosis . . . . . . . . . . . . . . . . . . . . . . . . . 38
21 SUVH9 −GFP during early prophase . . . . . . . . . . . . . . . . . . . . . . . 39
22 SUVH9 −GFP during prophase . . . . . . . . . . . . . . . . . . . . . . . . . . 40
23 SUVH9 −GFP during meiosis I . . . . . . . . . . . . . . . . . . . . . . . . . . 41
24 SUVH9 −GFP during meiosis II . . . . . . . . . . . . . . . . . . . . . . . . . . 41
25 Localization of rRNA genes . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
26 MBD6 −GFP xREC8 −mRUBY 3 during meiosis I . . . . . . . . . . . . . . . . . .
27 MBD6 −GFP xREC8 −mRUBY 3 during meiosis II . . . . . . . . . . . . . . . . . .
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28 Durations of meiotic stages . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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List of Tables
1 Standard PCR mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2 Liquid LB medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3 Solid LB medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4 S.O.C medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
5 Magic buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
6 Used antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
7 Table of used primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
8 Table of vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
9 Table of double constructs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
10 List of chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
11 Used equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
12 Ingredients for LR reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
13 Standard PCR cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
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1 Abstract
DNA methylation is an important epigenetic modification as it influences various cell functions
by controlling gene expression. Moreover, adaptions to environmental changes can be inherited to
future generations by alteration of DNA methylation. Little is known about the dynamics of DNA
methylation during meiosis so far. Here, the dynamics of the two novel reporter lines MBD6 −GFP
and SUVH9 −GFP for CG and CHH methylation respectively are investigated in detail during
meiosis by live cell imaging of male meiocytes in A. thaliana. During pre-meiosis, the global
DNA methylation is low and persists only in potentially centromeric and pericentromeric regions,
but an increase is observed during meiosis. Both reporters displayed a distinct pattern. In addition
to the methylation reporters, the meiocytes were simultaneously monitored with two meiotic
reporter lines: ASY3 or REC8. Microscope and co-localization analysis revealed an influence of
methylation on the formation of recombination events. Thus, the combination of meiotic and
methylation reporter line is a novel and useful approach to describe the progression through
meiosis and to understand the implication of methylation in this context better.
2 Zusammenfassung
DNA Methylierung ist eine bedeutsame epigenetische Modifikation, da sie durch Kontrolle der
Genexpression vielfätige Funktionen der Zelle beeinflusst. Des Weiteren können Anpassungen an
die Umwelt durch Vererbung veränderter DNA Methylierungsmuster an nachfolgende Generationen
weiter gegeben werden. Aktuell ist wenig bekannt über die Dynamik von DNA Methylierung
während der Meiose. Hier wurden die neuartigen Reporter MBD6 −GFP und SUVH9 −GFP für die
Detektion von CG beziehungsweise CHH Methylierung genutzt, um Methylierungsdynamiken
während der Meiose durch Lebendzellbeobachtung männlicher Meiozyten nachzuvollziehen.
Während der Prämeiose stellte sich die DNA Methylierung als gering heraus, es erfolgte jedoch
eine Zunahme im Laufe der Meiose. Beide Reporter zeigten ein spezifisches Muster. Ergänzend
zu den Methylierungsreportern wurden die Meiozyten zeitgleich mit zwei meiotischen Reportern
verfolgt: ASY3 oder REC8. Die Untersuchung durch Mikroskop- und Kolokalisationanalysen
enthüllten einen Einfluss der DNA Methylierung auf die Formation von Rekombinationsreignissen.
Daher ist die Kombination von meiotischen und Methylierungsreportern eine neuartige
und sinnvolle Herangehensweise um den Ablauf der Meiose zu beschreiben und vor diesem
Hintergrund den Einfluss von DNA Methylierung besser zu verstehen.
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3 Introduction
3.1 General function of DNA methylation
Function within the organism
Although all cells of an organism embody the same set of genomic material, gene expression
levels are tissue-specific and vary over time. This differential gene expression is crucial to maintain
the architecture of complex organisms where different type of cells needs to fulfil diverse
functions, requiring distinct and specific proteins. Thus, regulation of gene expression is the core
function within the cell and the entire organism. It controls not only the cell differentiation but
also governs the functions of the cell [Alberts et al., 2011a]. Epigenetic marks, such as DNA
methylation and histone modifications play an important role in the control of gene expression. A
required premise for gene expression is that the gene in charge lies within a region of loose chromatin
called euchromatin. Thus, accession of transcription activators is assured. Tightly packed
and transcriptionally silenced chromatin is called heterochromatin and affect pericentromeric
and telomeric regions or terminally differentiated cells [Graw, 2015a]. Chromatin condensation
is directly linked to epigenetic modifications. Histone modifications include acetylation,
phosphorylation, ubiquitination and mono-, di- or trimethylation, which lead, depending on
the placement, to chromatin compaction or loosening [Bannister and Kouzarides, 2011]. DNA
methylation does not only induce heterochromatin formation, but also controls gene expression
within euchromatin. Gene promoter methylation inactivates the respective gene as it hinders
transcription factors from binding. Promoter methylation is rather rare but seems to play an
important role in tissue-specific gene expression. In contrast, gene body methylation enhances
gene expression by so far unknown mechanisms [Zhang et al., 2006]. Since genomic regions
within heterochromatin are inactive, methylation-induced heterochromatin formation is important
for genomic imprinting and control of small interfering RNA (siRNA) producing regions
such as transposons [Zhang et al., 2006]. Transposon silencing is essential for genome stability.
Due to their ability to "jump" within the genome, transposable elements are always a "risk" as
they can copy themselves into different gene regions. This can lead to functional disruption
of the corresponding gene [Alberts et al., 2011b]. Epigenetic modifications of the genome are
not only useful to permanently change the transcriptional status of specific regions but also
for more short-term changes. By controlling gene transcription, the cell can adapt to different
environmental influences. As plants are stationary organisms, fluctuating adaption is the only
solution to fast changing environments [Mathieu et al., 2007].
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Inheritance of environmental influences
DNA methylation and other epigenetic marks do not only maintain phenotypic plasticity of the
organism itself but also make these adaptations heritable to future generations, which is the most
staggering feature of epigenetic regulation. This effect is known as adaptive transgenerational
plasticity and serves the purpose to improve the offspring’s adaptation to the current environment.
The parents inherit an epigenetic change to their progeny that is modified according to the
conditions they experienced. This adaptation induces for example a higher stress tolerance.
Adaptive stress-responses include abiotic stresses such as drought, heat or nutrient stress but
also biotic stress like pathogen defense and herbivore damage. Inherited adaptations are passed
on from maternal plants to the offspring via seed provisioning or storage of mRNAs, hormones
and proteins in the seeds. Adaptations can also be transmitted by histone modifications and
DNA methylation, persisting in multiple generations [Herman and Sultan, 2011]. Most abiotic
and biotic stresses trigger a global DNA methylation and homologous recombination frequency
(HRF) increase in the offspring. This will induce greater genetic variability in the resulting
progeny, even if the offspring lives in non-stress conditions [Boyko et al., 2010].
3.2 Establishment, maintenance and function of DNA methylation
DNA methylation is the attachment of a methylgroup to a cytosine producing 5-methylcytosine.
In plants, cytosines in a CG, CHG or CHH context (H=A,T or C) can be methylated [Chan et al., 2005].
In most cases, this modification has a repressive effect. Furthermore, methylation is also
found in gene bodies of highly expressed genes and thus hypothetically enhances transcription
[Zilberman et al., 2007]. The different contexts are established and maintained by different
pathways, probably due to their differential function.
RdDM pathway
Guidance of proteins by small interfering RNAs is an important function within the cell in order
to modify transcriptional regulation at specific target regions. This does not only play a role in the
post-transcriptionally silencing RNA interference pathway (RNAi) but also in the RNA-directed
DNA methylation pathway (RdDM). Here, the methyltransferase DOMAINS REARRANGED
METHYLTRANSFERASE 2 (DRM2) is guided by 24-nucleotide (nt) siRNAs to establish de
novo DNA methylation in CG, CHG and CHH contexts at applicable siRNA-producing sites like
transposons.
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Figure 1: RNA-directed DNA methylation pathway. The mechanisms of the RdDM pathway
are complex and largely hypothesized. It is proposed that Pol IV produces ssRNA which is in
turned used by RDR2 to generate dsRNA. DLC3 uses these transcripts to produce 24-nt siRNA
that are then loaded onto AGO4. Overall multiple interactions, DRM2 in then recruited to sites
where AGO4 and other components are present. Figure from [Law and Jacobsen, 2010].
Due to its complexity, the exact mechanism and proteins implicated in the RdDM pathway
remain widely unclear. This model connects most so far available data. The biogenesis of the
24-nt siRNAs starts with the production of single stranded RNA transcripts corresponding to
transposons and other repetitive elements mediated by RNA polymerase IV (Pol IV). RNA-
DEPENDENT RNA POLYMERASE 2 (RDR2) generates double stranded RNAs from these
transcripts, followed by the procession of 24-nt siRNAs by DICER-LIKE 3 (DLC3). The 24-nt
siRNA can be loaded to Argonaute protein 4 (AGO4). AGO4 interacts with two subunits of the
RNA polymerase Pol V, NUCLEAR RNA POLYMERASE E1 (NRPE1) and NRPE2, as well
as DRM2. Pol V generates intergenic non-coding region (IGN) transcripts, which serve as a
scaffold to recruit AGO4. AGO4 can also interact with SUPPRESSOR OF TY INSERTION
5-LIKE (SPTL5). This association bridges siRNA and IGN producing sides via the interference
of AGO4-bound siRNAs and IGNs. Another protein, INVOLVED IN DE NOVO2 (IDN2) will
recognize this interactions und thus recruit DRM2 only to sites that produce both transcripts (see
Fig. 1). RdDM is the main pathway to establish de novo DNA methylation at CG, CHG and
CHH context and thus to induce heterochromatin formation [Law and Jacobsen, 2010].
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Maintenance and function of CG methylation
CG methylation is the most frequent methylation context and is maintained by the methyltransferase
METHYLTRANSFERASE 1 (MET1), the methylcytosine-binding proteins of the
VARIANT IN METHYLATION (VIM) family and DEFICIENT IN DNA METHYLATION
1 (DDM1), an ATPase and nucleosome remodeler of the SWI2/SNF2 family. In brief, newly
synthesized DNA strands lack methylation, after DNA replication leaves previously symmetrically
methylated CG sites hemimethylated, a circumstance that VIM proteins can detect via their
mCG-binding SRA-domain. MET1 is then recruited by VIM and reestablishes the fully methylated
state. DDM1 plays an essential role in maintaining CG methylation in heterochromatic
transposable elements (TE) by making the chromatin accessible to MET1 and by overcoming
the inhibiting effect of the linker histone H1. In euchromatin on the other hand, DDM1 is not a
requirement for chromatin accessibility [Zemach et al., 2013].
In plants, heritable and long-term adaptations are facilitated by CG methylation [Mathieu et
al., 2007]. Thus, the plant uses CG methylation for genomic imprinting, transposon silencing,
cell differentiation, tissue-specific gene expression and promoter and gene body methylation.
[Chan et al., 2005, Song et al., 2005, To et al., 2015].
Maintenance and function of non-CG methylation
The maintenance of symmetrical CHG methylation is assured by a reinforcing loop between
DNA methylation and histone 3 lysine 9 dimethylation (H3K9me2). This pathway involves the
CHG methyltransferase CHROMOMETHYLASE 3 (CMT3) and the histone methyltransferase
SUPPRESOR OF VARIEGATION 3-9 HOMOLOGUE 4 (SUVH4, also known as KYP), which
is responsible for H3K9 dimethylation (H3K9me2). Two other histone methyltransferases,
SUVH5 and SUVH6 also play a role in the global CHG methylation. CMT3 cannot only
methylate DNA but also binds methylated histone H3 tails with its chromodomain. Thereby,
it is recruited by histone 3 lysine 9 (H3K9) mono-, di- or trimethylation. SUVH4 on the
other hand consists of a histone methylation domain and an SRA domain that can bind CHG
methylation specifically. CMT3 also potentially controls CHH methylation at some loci. Recently,
another chromomethylase, CMT2, was discovered. CMT2 plays a role in CHG and CHH
maintenance, using the same reinforcing loop as CMT3 and thus working as a redundant CHG
methyltransferase to CMT3 [Hume Stroud et al., 2014]. Both CMT2 and CMT3 need DDM1 to
grant them access to the heterochromatin, similar as for MET1 in CG methylation maintenance
[Zemach et al., 2013]. CHH methylation is not only maintained by CMT2 and CMT3 but the
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DRM2 methyltransferase contributes as well by constant de novo methylation by the RdDM
pathway. On the contrary, DRM2 only plays a minor role in mCHG maintenance and has no
effect on mCG preservation [Cao et al., 2003]. The SUVH9 protein, containing an SRA-domain,
works within the RdDM pathway and may play a role to conserve DRM2 activity at asymmetric
CHH sites [Johnson et al., 2008]. Non-CG methylation is exclusive to the plant kingdom and
most likely evolved as a redundant pathway to ensure transposon silencing. Generally, non-CG
methylation is less stable than CG methylation and it is not inherited [Dalakouras et al., 2012].
Besides transposon silencing, it rather has a role in immediate, non-heritable stress responses
[Mathieu et al., 2007]. Non-CG methylation can also be found in silent gene promoters and thus
influence gene expression [Zhang et al., 2006].
Mechanisms of Demethylation
Mechanisms of demethylation are crucial to guarantee variable gene activation. Demethylation
occurs passively by loss of methylation during replication or actively by removal of methylated
cytosines [Law and Jacobsen, 2010]. Active demethylation in A. thaliana is carried out by the
DNA glycosylases REPRESSOR OF SILENCING 1 (ROS1), DEMETER (DME), DEMETER-
LIKE 2 (DML2) and DML3. All of these DNA glycosylases recognize methylated cytosines in a
sequence independent manner and remove them by breaking the N-glycosidic bond and base
excising at the DNA backbone. The repair of the DNA gap is carried out by a so far unknown
DNA polymerase and lyase, but it is proposed that the base excision repair (BER) pathway is
implicated. The process by which ROS1, DME, DML2 and DML3 find their specific target loci
remains yet to be explored. In contrast to other DNA glycosylases, DME is specifically expressed
in the central cell of the female gametophyte to establish imprinting during gametogenesis. Plants
initiate imprinting by the removal of their respective methylations. DML2, DML3 and ROS1
function redundantly in vegetative tissues such as roots and leaves. It is not yet fully understood
what exactly their function is, but it is suggested that they protect genes from being methylated.
These genes are close to heterochromatic regions and thus targeted by RdDM. Furthermore, they
keep transposons in an adaptive but silenced state to enable reactivation during gametogenesis.
Passive demethylation occurs during replication when the methyltransferases that are responsible
for remethylation are not expressed. This process is observed in the endosperm of the female
gametophyte where MET1 activity is suppressed to establish imprinting [Jullien et al., 2008].
It is thought that passive demethylation works together with active demethylation as it aids
DME function. A lack of MET1 results in hemimethylated DNA which improves DME activity
and reduces the risk of DNA double strand break. The decrease of MET1 can also inhibit
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Figure 2: Structures of CMT3 and CMT2. Top. Structure of CMT3 with highlighted chromodomain
(A) and S-adenosyl-L-methionine-dependent methyl transferase (B). Bottom. Structure
of CMT2 with highlighted chromodomain (C) and S-adenosyl-L-methionine-dependent
methyl transferase (D). Figure from SWISS-MODEL [Biasini et al., 2014, Kiefer et al., 2008,
Arnold et al., 2006, Guex et al., 2009].
instantaneous remethylation of newly demethylated DNA [Law and Jacobsen, 2010].
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3.3 Methylation during Meiosis
Principles of Meiosis
Meiosis is constituted of two consecutive divisions with the first one being a reductional division
as the ploidy is reduced from diploid to haploid. The second division is equatorial, similar
to a mitosis, in which the sister chromatids are segregated leading to four haploid daughter
cells. Cyclin-dependent kinases (CDKs) are the main regulators of the cell cycle and meiotic
progression. Throughout the cell cycle different proteins are specifically phosphorylated by
CDKs in specific time points, making the right timing of CDK activity crucial for the cell cycle
progression. CDKs can interact with activating cyclins and inhibiting KIP-RELATED PRO-
TEINS (KRP). The CDK-cyclin complexes can mainly be inactivated by P-loop phosphorylation
or activated by phosphorylation of their T-loop by CDK-activating kinases (CAKs). Different
CDK mutants have been generated and described in order to better understand their role during
cell cycle and meiosis [Dissmeyer et al., 2009]. Another important protein that works within the
cell cycle control is RBR1-2. This protein is required for correct cell proliferation, including cell
division and differentiation in gametic and accessory cell types [Johnston and Gruissem, 2009].
Meiosis I starts with the alignment of homologous chromosomes in prophase I. In early prophase,
during leptotene, the chromosomes start to condense, resulting in visibly condensed regions
called chromocenters. Furthermore, as chromosome synapsis starts, the induction of double
strand breaks will result in crossing-over formation later on. Proteins of the meiotic cohesion
complex such as REC8 are important throughout prophase I, as they mediate the sister chromatid
cohesion and chromosome organization [Yoon et al., 2016]. Another protein involved
in sister chromatid and centromere cohesion is DYAD/SWI1, which mediates bivalent formation
[Agashe et al., 2002]. The chromosome condensation and synapsis further proceeds in
zygotene and the reparation of non-crossing over double strand breaks starts. The homologous
chromosomes begin to align in several spots due to the formation of the synaptonemal
complex, consisting of axial-element associated proteins such as ASY1, ASY3 and ZYP1.
[Ferdous et al., 2012]. In pachytene, the homologous chromosomes are fully aligned and connected
by the synaptonemal complex. Additionally, crossing-overs are present at previously
induced and not repaired double strand breaks. During the last stage of prophase I, diakinesis,
the synaptonemal complex disappears and the bivalents stay connected only in chiasmata regions.
Chiasmata indicate regions of recombination, produced by crossing-overs. In metaphase I, the
bivalents align in the equatorial plane and the spindle apparatus, constituted of microtubules,
separates the five bivalents. In anaphase I, the homologous chromosomes segregate to the
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opposite poles of the cell. Then the two pools of chromosomes decondense and the organelle
band separates them from one another during telophase I. Directly after, the chromatin condenses
again in prophase II, followed by the alignment of the chromosomes in the equatorial plane
during metaphase II. The sister chromatids are then separated in anaphase II. Once at the opposite
poles of the cell, the chromatids decondense in telophase II and the tetrade splits, forming four
haploid spores [Ross et al., 1996, Graw, 2015b].
Influences of methylation on recombination
Besides the reduction of the genetic content, recombination is another important process that
occurs during meiosis. The frequency of recombination events is not steady within the genome
as there are so called "crossing-over hot spots" where recombination occurs more frequently.
The distribution of crossing-overs along the chromosomes is not random, depending on interference
and homeostasis. Interference is the reciprocal influence that crossing-over hot
spots have on their localization to each other, while homeostasis describes the appearance of
crossing-over hot spots on a stable frequency, independent on double stand break frequency
[Higgins et al., 2004, Martini et al., 2006]. Crossing-over hot spots depend on the histone variant
H2A.Z and correlate with transcriptional start and termination sites, as well as accessible
chromatin marks such as H3K4me3, low DNA methylation and low nucleosome density, represented
by a low nucleosome occupation of the DNA [Choi et al., 2013]. High DNA methlyation,
histone marks such as H3K9me2 and high nucleosome density on the other hand suppress recombination
hot spots [Yelina et al., 2015]. This leads to a variable crossing-over distribution along
the chromosomes. Heterochromatic regions, rich in transposons and repetitive elements, are
usually heavily methylated and thus recombination suppressed, for example at the centromeres.
Gene-rich regions on the other hand are within open chromatin and hence show an increased
crossing-over frequency [Drouaud et al., 2007]. Loss of DNA methylation is sufficient to initiate
remodeling of crossing-over hot spots, although the overall frequency of recombination events
remain similar. An increase in centromeric recombination is related to a severe methylation
loss in this region. Furthermore, pericentromeric crossing-overs are decreased and hot spots in
euchromatic chromosome arms increased, probably due to homeostasis. Nevertheless, the exact
reason and mechanisms remain to be uncovered [Yelina et al., 2012, Mirouze et al., 2012].
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Chromatin dynamics during premeiosis
In plants, germline cells are established from and within somatic cells in floral organs in
late development. Spore mother cells (SMCs) specialize from somatic precursor cells and by
undergoing meiosis and two rounds of mitosis they form the multicellular gametophyte. This
differentiation is accompanied by global chromatin reorganization, at least in the female SMCs.
The depletion of H1 linker histones is one of the earliest signs of megaspore mother cell (MMC)
differentiation. It comes along with global nucleosome remodeling, chromatin decondensation
and loss of heterochromatin. The chromatin is shifted to a more active state, supported by the
change in histone marks. The activating histone mark H3K4me3 is increased in MMCs while
repressing H4K27me1, H3K27me3 and H3K9me1 decrease. The chromatin remodeling and
global loss of heterochromatin may serve different purposes. First, SMC differentiation results
from a change in gene expression due to the exit of the SMC from the somatic program. To
undergo meiosis, meiotic genes have to be activated. It is indicated, that the RdDM pathway
plays a role in the transition from somatic to meiotic program. Second, the spore cells need to be
in a pluripotent state to form a multicellular gametophyte. Thus, the cell undergoes epigenetic
reprogramming, meaning that epigenetic marks corresponding to the previous somatic cell
function are erased. However, the methylome landscape was not observed in this context so far.
The loss of heterochromatin also leads to TE reactivation in SMCs, although they seem to be
contained by the RdDM pathway [She and Baroux, 2014].
3.4 Visualization of Methylation - DYNAMET reporters
Two reporter proteins were recently developed by Ingouff et al., (2017) to observe the changes
of methylation patterns during gamete development by live cell imaging. To achieve this aim,
two proteins with specific methylation-binding domains were used. One reporter targeted
symmetrical CG methylation and the other asymmetrical CHH methylation.
Symmetrical mCG - MBD6-GFP
The A.thaliana protein METHYL-CpG-BINDING DOMAIN-CONTAINING PROTEIN 6
(MBD6) possesses a methyl-binding-domain (MBD) that aims at symmetrical mCG. Once
bound to mCG, MBD6 recruits histone deactylases and chromatin remodeling factors leading
to chromatin compaction and hence transcriptional silencing. Thus, MBD6 works as a
15

mediator between DNA methylation and chromatin state [Zemach and Grafi, 2003]. The MBDdomain
alone is sufficient to bind methylated CG and was therefore used for the reporter design
[Ingouff et al., 2017, Zemach and Grafi, 2003].
The resulting reporter binds specifically to methylated CG sites and also accumulates at highly
methylated transposable elements. As it also enriches at low methylated sites such as gene
bodies, it cannot be used to measure the quantity of methylation [Ingouff et al., 2017].
Figure 3: mCG-Venus during male sporogenesis. Signal of original DYNAMET mCG-Venus
as designed by Ingouff et al.,(2017).
Asymmetrical mCHH - SUVH9-GFP
The SUVH9 protein binds specifically to CHH methylation and plays a role in the RdDM
pathway of mCHH maintenance. Two domains of SUVH9 are of major importance: the
methylcytosinebinding domain SRA and the SET domain, which aligns closest to known histone
methyltransferasedomains, hinting a function within histone methylation. Nevertheless, the
precise function of this domain remains to be uncovered as no histone methyltransferase activity
was observed yet. The exact function of SUVH9 within the RdDM pathway is currently unknown
but it is proposed that it may retain DMR2 at recently methylated sites or recruits an unknown
component that is needed for DMR2 activity [Johnson et al., 2008]. The SRA-domain alone is
not sufficient to bind methylated sites, therefore the whole SUVH9 protein was used to design
the reporter. The final reporter binds mCHH in a highly specific and quantitative way. Hence it
can be used as a quantitative measurement of methylation [Ingouff et al., 2017].
16

Figure 4: mCHH-Venus during male sporogenesis. Signal of original DYNAMET mCHH-
Venus as designed by Ingouff et al., (2017).
3.5 Aim of this project
Due to the lack of appropriate reporter proteins, observation of DNA methylation dynamics
during meiosis with live cell imaging will be a novel approach. Thus, the prior aim of this
project will be to test whether and to what extent male meiosis can be monitored with the
above-mentioned reporters. Of special interest will be where and when methylation is present
during pre-meiosis and meiosis. Furthermore, the observation of the overall dynamics throughout
the meiotic cell division will be explored in detail. Due to the different binding-preferences of
the two reporters, possible differences between CG and non-CG methylation will be important to
analyse as well. The main question of interest will be to interpret and discuss these findings, in
case of potential relevant information for recombination, gene expression or transposon silencing
during male meiosis.
17

4 Material and Methods
4.1 Material
Plant material
All plants were from Columbio-0 ecotype. Seeds were put on 1/2 MS plates and into 21 ◦ C /
18 ◦ C, 16 hour daylight light chambers with 50 % humidity for initial growth. After a week of
growth, seedlings were then put on a 3:2:1 mix of soil, sand and granulate. The plants were kept
in light chambers with 16 hour day cycle with 21 ◦ C, 67% humidity and 104 µmol illumination
until ready for use.
PCR mix
Mix used for all polymerase chain reactions.
Table 1: Standard PCR mix. Volume of Ingredients used for PCR mixes. Volumes are listed
per 25 µl well.
Ingredient
Volume [µl]
Water 10.5
DreamTaq Green PCR master mix 12.5
Forward Primer 0.5
Reverse Primer 0.5
Template DNA 1
Agarose gel
For gel electrophoresis, 3g of agarose were mixed with 300ml TE buffer and heated to obtain a
1% agarose gel.
1/2 Murashige-Skoog medium
For seed inoculation, 1/2 MS medium was used. Per one liter OF water, 5g sucrose and 2.2g
Basal salts were added and brought to ph=5.8. Per 250ml solution 2.5g agarose was added into a
Schott bottle. The glass bottles were autoclaved and reheated for usage.
18

LB medium
Table 2: Liquid LB medium. Ingredients and Volumes for 1l liquid LB medium.
Ingredient
Trypton
Yeast-extract
NaCl
Water
Volume
10g
5g
5g
1l
Table 3: Solid LB medium. Ingredients and volumes for 1l solid LB medium.
Ingredient
Trypton
Yeast-Extract
NaCl
Water
Agar-Agar
Volume
10g
5g
5g
1l
16g
S.O.C. medium
Table 4: S.O.C. medium
Concentration Ingredient
2.0 % Tryptone
0.5 % Yeast-Extract
10 mM Sodium chloride
2.5 mM Kalium chloride
10 mM Magnesium chloride
10 mM Magnesium sulfate
20 mM Glucose
Magic buffer
Table 5: Magic buffer. Ingredients for Magic buffer used for DNA extraction.
Ingredient Concentration [mM] Mass per liter[g]
Tris-HCl 150 6.057
NaCl 300 17.532
Sucrose 300 102.69
19

Used antibiotics
Table 6: Used antibiotics. List of used antibiotics with corresponding type of usage
Antibiotic
Spectomycin
Hygromycin
Carbenicilin
Usage
Selection for transformed E.coli and A. tumefaciens
Selection for transformed A. thaliana seedlings
Selection for transformed A. thaliana seedlings
Primer sequences
Primers were sent to production to Sigma-Aldrich Co.
Table 7: Table of used primers. Primers used to check for successful LR reaction.
Primer
GFP_300F
GFP_300R
TagRFP_200F
TagRFP_200R
mRUBY3_250F
mRUBY3_250R
Sequence
GAAGGGCATCGACTTCAAGG
TTGAAGTCGATGCCCTTCAG
CCTGATCTGCAACTTCAAGACCAC
CATGAAGCTGGTAGCCAGGATGTC
GAACACGGAGATGATGTATCCAGC
TGGGAATGACTGCTTAAAGAAGTC
Double constructs
Each double construct consisted of a methylation and a meiosis gene. Both methylation reporters
were combined with one of the two meiotic reporters and a centromeric reporter, resulting in
six different double constructs (Tab. 9). The double constructions were produced by Multisite
Gateway Three-Fragment Vector construction LR reaction using the vectors of table 8. The entry
vectors derived from BP reactions with the pDONRp4p1r and pDONR221 vectors.
All pDONR and destination vectors came with the kit used for Multisite Gateway Three-Fragment
vector construciton.
Sequences for every vector can be found in the supplementaries.
Table 8: Table of vectors. Vectors used for LR reaction to establish double constructs.
Number Vector
1 pENTR2B-pHTR5-MBD-NLS-GFP 3’UTR
2 pENTR2B-pHTR-SUVH9-NLS-GFP 3’UTR
3 pENTR-PROREC8-REC8-mRUBY3 3’UTR
4 pENTR-PROCENH3-TagRFP-CENH3 3’UTR
5 pENTR-PROASY3-gASY3-TagRFP 3’UTR
Besides the vectors in table 8, the destination vector R4pGwB501 was used.
20

Table 9: Table of double constructs. Methylation reporter (top line) and meiotic reporter (first
column) used for design of double constructs.
Reporter MBD6-GFP SUVH9-GFP
ASY3-RFP MBD6-GFPx ASY3-RFP SUVH9-GFPxASY3-RFP
REC8-mRUBY MBD6-GFpxREC8-mRUBY SUVH9-GFPxREC8-mRUBY
CENH3-RFP MBD6-GFPxCENH3-RFP SUVH9-GFPxCENH3-RFP
List of chemicals
Table 10: Chemicals. List of used chemicals and manufactures.
Chemical
Sucrose crystallized
Agarose powdered food grade
Murashige & Skoog Medium Basal salt mixture
Neudomück
Tryptone
Yeast extract
Sodium chloride
TRIS
EDTA
Hydrochloric acid 37 %
Potassium chloride
Magnesium chloride
Magnesium sulfate
Glucose
Manufacture
Duchefa Biochemie
AppliChem
Duchefa Biochemie
Progema
Duchefa Biochemie
Duchefa Biochemie
Duchefa Biochemie
Duchefa Biochemie
VWR chemicals
VWR chemicals
Merck
Applichem
Applichem
Duchefa Biochemie
21

Equipment list
Table 11: Used equipment.
Equipment Product/Company
Pipettes
Eppendorf research plus
Pipette tips Sarstedt
Tubes 1.5 ml Eppendorf
Falcon tubes 50ml Sarstedt
PCR cycler Biometra Tadvanced
Biometra Tprofessional
Thermo cycler Eppendorf ThermoMixer C
Mixer mill Retsch Mixer Mill M300
PCR wells Sarstedt
Starlab
Gel chamber Thermo Fisher Scientif Owl EasyCast B1A/B2
Peqlab
Angewandte Gentechnologie Systeme GmbH
Binocular Zeiss Stemi 508
SZ-ST Olympus
Microscope Zeiss LSM 880 with Airyscan
4.2 Molecular methods
Multisite Gateway Three-Fragment Vector construction
Multisite Gateway Three-Fragment Vector construction relies on a bacterial recombination
system. This recombinatory system consists of four recombination sites, called attB, attP, attR
and attL, whereby attB and attP or attR and attL respectively can recombine with each other. If
the recombination site lies downstream of the gene of interest, thus pointing away the gene, the
site gets the appendage "r". The recombination of attB/attBr and attP/attPr result in a attL or
attR site respectively. attL and attR sites produce a attB site (see Fig. 5).
The Multisite Gateway Three-Fragment Vector construction was performed according to the
protocol handed in with the kit. The mix for the LR reaction is shown in table 12. All vectors
and enzyme mixes used for the Multisite Gateway Three-fragment vector construction came
with the respective kit by Invitrogen.
22

Figure 5: Multisite Gateway Three-Fragment Vector construction According to the protocol
by Invitrogen - Life Technologies [Invitrogen, 2012]. Since a double construct is supposed to
be produced, the Multisite Gateway reaction was carried out using two fragments. The PCR
fragments are cloned into pDONOR vectors using a BP reaction, resulting in the two entry
clones. A LR reaction is used to recombine the genes into the destination vector, producing the
expression clone. Figure from [Invitrogen, 2012].
Table 12: LR reaction. Mix for Multisite Gateway Three-Fragment LR reaction.
Ingredient Volume µl
each entry vector 1
destination vector 1
TE buffer 2.5
LR Clonase II 0.5
Plasmid extraction
Plasmids were extracted from Escherichia coli with the GeneAid Mini Plasmid Kit. The kit was
used according to the instructions handed in with the kit.
23

Transformation check
All transformations of Escherichia coli were checked by PCR using the standard PCR mix
(Tab. 1) and PCR cycle () with corresponding primer pairs according to table 7 and ruand a gel
electrophoresis on 1% agarose gel. The gels were run on 120V for 30-45mins.
Table 13: PCR cycle. Standard PCR cycle used for all PCRs.
Step Temperature Duration
1 95 ◦ C 2 min
2 95 ◦ C 30 sec
3 55 ◦ C 30 sec
4 72 ◦ C 1 min
5 72 ◦ C 10 min
6 4 ◦ C ∞
Steps 2-4 were repeated 30 times.
DNA extraction
To extract DNA from leaves, 250 µl Magic buffer, one bead and one leaf were put into each well
of a deep well-plate. It was then shaked for 5 minutes at a mixer mill at 25 1/s. 50 µl of the DNA
extract is transferred from the deep well-plate to new PCR DNA well and kept in the freezer at
-40 ◦ C.
Seed sterilization
Seeds for sterilization were filled into eppendorf tubes on a rack and put into a vacuum desiccator.
The lids were opened. A 50ml beaker was filled with 30ml bleach (7.5 %) and 1 ml HCl and put
next to the rack . The desiccator was closed and a vacuum produced. The seeds remained in the
desiccator about three hours. The lids were then closed and the rack put on a clean bench with
opened lids for at least 20-30 mins.
Plant screening
Plant seeds were screened for transgenic plants by using 1/2 MS medium with 0.015% Hygromycin
and 0.05 % Carbenicilin.
24

4.3 Transformation
Transformation of Escherichia coli
5 µl of plasmid DNA was added to One shot of TOP10 chemically competent E.Coli carrying
Kanamycin resistance. It was then incubated on ice for 30 minutes, followed by a heat shock
of 42 ◦ C for 30 seconds. Afterwards, the bacteria were placed on ice for 2 minutes. 250 µl of
S0.O.C. medium was pre-warmed and added to the bacteria. The mix was shaken at 725 rpm
for 1 hour at 37 ◦ C and then spread on a 0.1 % Spectomycin selection plate prior to incubate
overnight incubation 37 ◦ C.
Transformation of Agrobacterium tumefaciens
1 µg of extracted plasmid were added to frozen competent A. tumefaciens and incubated for
5 min at 37 ◦ C. After 2 min the mix was gently mixed. Immediately after heat incubation, the
sample was incubated on ice for 15 - 30 mins. 1 ml of LB medium was added and the mix was
incubated at 28 ◦ C for 1 - 3 hours. Afterwards the culture was centrifuged at maximum speed for
1 min, followed by the resuspension of the pellet in 100 µl LB medium. The culture was plated
on an LB selection plate with 0.1 % Spectomycin and incubated at 28 ◦ C for 2 days.
Glycerol stock of A.tumefaciens
For a better storage, a glycerol stock of A. tumefaciens was made. Afterwards, one colony was
picked from the LB selection plate and then transferred into a tube with 3 ml LB medium and
0.1 % Spectomycin. The culture was incubated at 28 ◦ C overnight. 700 µl of the grown culture
was mixed with 300 µl glycerol and stored on -80 ◦ until further use.
Transformation of Arabidopsis thaliana
Liquid culture To transform Arabidopsis thaliana, a liquid culture of the transgenic Agrobacterium
tumefaciens glycerol stock was used. A toothpick was dipped into the glycerol stock and
put into a tube with LB medium and 0.1 % Spectomycin. The culture was incubated for 24 hours
at 28 ◦ C. After incubation, 500 µl of the bacteria culture was transferred in a 100ml LB medium
with 0.1% Spectomycin. The mix was again incubated at 28 ◦ C overnight.
25

Floral Dip To transform Arabidopsis thaliana, a variation of the method "Floral Dip" was used
[Clough and Bent, 1998]. According to the protocol, all siliques and open flowers of the plants
were cut. The grown bacteria on the liquid A. tumefaciens culture was centrifuged at 4000rpm
for 5 minutes at room temperature. The supernatant was discarded. The pellet was resuspended
in a water solution consisting of 300ml water, 15g crystallized sucrose and 200 µl Silwett. The
prepared plants were fully immersed into the water solution for 10 seconds and put into darkness
for 24 - 48 hours.
4.4 Confocal microscopy
The microscope analysis of all transgenic A. thaliana plants was done with the Zeiss LSM 880
with the Airyscan Unit. This microscope is equipped with a Argon Laser containing the exitation
wave length for GFP at 488nm, Diode Laser (DPSS) for the RFP exitation at 561 nm and a
Helium-Neon to collect the autofluorescence at 633nm. For image acquisition and processing
the software ZEN black was used. Excitation laser powers were kept between 2% and 3% during
the acquisition to minimize photo bleaching. The time lapse interval used was 10 minutes during
prophase I and 5-8 minutes after metaphase I. For the image acquisition two seperate channels
were used and two different detectors assured the correct separation of the GFP emission from
the RFP and autofluorescence.
Signal checking
Before the live cell imaging experiment, the transgene expression in the plants were checked
for proper protein localization. For that purpose, a bunch of flower buds was taken in whole
and sorted by size (Fig. 6 left) . Flower buds of the right size were opened, petals and sepals
removed. The anthers were transferred to a water drop on a microscope slide and covered with a
coverslip. For image acquisition, the 40x1.0DIC objective was used.
Live cell imaging
For live cell imaging a bunch of flower buds containing the stem were removed from the plant.
One flower bud of the right size was chosen and the others were removed from the stem. The
outer petal was dissected from the remaining flower bud and the sample was transferred to a
small petri dish with 1% agar and 1/2 MS medium. The sample was then fixed with a drop of
26

1% Agarose without covering the anthers themselves. For acquisition, the petri dish was filled
with water (Fig. 6 right) and the 40x1.0DIC water immersion objective was used.
Figure 6: Sample preparation. Left: Orientation for anther size collection. For male live cell
imaging, the highlighted anthers are in the right stages. Right: Anther preparation for live cell
imaging. Picture provided by Maria Prusicki.
27

5 Results
The prior aim of this study is to monitor the two DNA methylation reporter lines MBD6 −GFP and
SUVH9 −GFP to follow DNA methylation dynamics during male meiosis. For this purpose, each
methylation reporter line was combined with a meiotic reporter: ASY3 −TagRFP or REC8 −mRUBY 3
by using Multisite Gateway Three-Fragment vector construction as described in the material
and methods. After successful transformation of the A. thaliana Columbia-0 plants with these
double constructs, the T 1 seeds were collected, sterilized, screened and the seedlings put on soil.
After three to four weeks of growth under controlled conditions, the plants were used for live
cell imaging.
5.1 Detection of CG methylation with MBD6 −GFP
Fluorescent reporter lines are one of the most useful tools for investigating dynamic protein
localization over time in living organisms. The combination of several reporter lines is crucial
to understand and describe the methylation pattern during meiosis. For this purpose, the
symmetrical CG methylation reporter line MBD6 −GFP was combined with the meiotic reporter
line ASY3 −TagRFP or REC8 −mRUBY 3 . The meiotic stage was defined by the cell shape and the
meiotic reporters ASY3 −TagRFP or REC8 −mRUBY 3 .
During pre-meiosis, the meiocyte cell shape is very squared, the nucleus visible in the bright field
and the nucleolus central. During this stage, ASY3 −TagRFP is not expressed yet, REC8 −mRUBY 3
shows a very diffuse signal throughout the nucleus and more interestingly, MBD6 −GFP is
specifically expressed in several spots localized near the nuclear envelope (Fig. 7, Fig. 8). We hypothesized
that these dots could be centromeric or telomeric regions. Therefore, the centromeric
reporter line TagRFP− CENH3 was used in addition to MBD6 −GFP to study the colocalization of
these proteins (Fig. 9). The layers of tapetum cells around the meiocytes show a similar pattern
but their methylation level is higher compared to the meiocytes (Fig. 8 ).
28

Figure 7: MBD6 −GFP during pre-meiosis. Signal of MBD6 −GFP (A) before the start of
meiosis. Premeiosis is indicated by the squared shape of cells (B). The detected methylation is
low, compared to surrounding somatic cells. MBD6 −GFP is localized at the nuclear envelope
(C).
Figure 8: MBD6 −GFP xREC8 −mRUBY 3 during pre-meiosis. Premeiosis can be defined by global
REC8 −mRUBY 3 distribution in the nucleus, central position of the nucleolus (A) and squared cell
shape (C). The dots of MBD6 −GFP indicate methylated regions (B). MBD6 −GFP is localized
near the nuclear envelope (D).
Observation of MBD6 −GFP and TagRFP− CENH3 revealed partial colocalization of the proteins
(Fig. 9 E: Pearson’s R=0.61 ) as indicated by a yellow merged signal (Fig.9 Top right). The
analysis of signal intensities in overlapping regions (white square) shows a strong colocalization
(Fig 9 Bottom right). Some CG methylation can be found apart from centromeres.
29

Figure 9: MBD6 −GFP x TagRFP− CENH3 in DE +/− during pre-meiosis. Top right:
MBD6 −GFP and TagRFP− CENH3 partially colocalize during pre-meiosis. Bottom right: The
signal intensities of colocalizing regions (square) peak around the same regions. Left: (E) 2D
histogram of MBD6 −GFP and TagRFP− CENH3 partial overlap. Pearson’s R=0.61, Mander’s M1=
0.754, Mander’s M2=0.75.
In early meiosis, the axial element of the synaptonemal complex ASY3 −TagRFP starts its expression
and localizes in the nucleus as well as REC8 −mRUBY 3 , which starts to show a specific
localization at the chromosomes (thin strands). Furthermore, the nucleolus is not central anymore
but lateral and close to the nuclear envelope. The nucleolar signal of MBD6 −GFP is diffuse and
thus different from the nuclear MBD6 −GFP . Generally, the level of MBD6 −GFP increases as
previously observed dots extent to thin threads along the chromosomes. Although the expression
of all three proteins increased, MBD6 −GFP shows no colocalization with ASY3 −TagRFP or
REC8 −mRUBY 3 (Fig. 10).
30

Figure 10: MBD6 −GFP xREC8 −mRUBY 3 or ASY3 −TagRFP during early prophase. A:
MBD6 −GFP xREC8 −mRUBY 3 show the lateral positioning of the nucleolus and thin strands of
chromosomes. B: MBD6 −GFP xASY3 −TagRFP displays a global signal of ASY3 (left). Both
show an extension of methylation by short threads (red arrows A: left, B: middle). The tapetum
cells are highly methylated compared to the meiocytes (white arrow).
Further condensation of the chromosomes in early to mid-prophase is displayed by thickening
and extending strands of ASY3 −TagRFP and REC8 −mRUBY 3 . The expression of MBD6 −GFP
increases likewise, indicated by the expansion of methylated spots as well as lengthening of
threads. Moreover, REC8 −mRUBY 3 colocalizes with MBD6 −GFP (Fig. 11) while ASY3 −TagRFP
does not (Fig. 12A, B: right).
31

Figure 11: Colocalization of MBD6 −GFP with REC8 −mRUBY 3 and ASY3 −TagRFP . Left: 2D
histogram of the partial overlap of MBD6 −GFP and REC8 −mRUBY 3 during early to mid-prophase.
Pearson’s R=0.71, Mander’s M1= 0.377, Mander’s M2= 0.59. Middle: 2D histogram of the
partial overlap of MBD6 −GFP and REC8 −mRUBY 3 during mid-prophase. Pearson’s R= 0.6,
Mander’s M1= 0.195, Mander’s M2= 0.51. Right: 2D histogram of the partial overlap of
MBD6 −GFP with ASY3 −TagRFP during the middle to end of prophase. Pearson’s R=0.64,
Mander’s M1= 0.728, Mander’s M2= 0.459.
The process of further chromosome condensation and alignment of ASY3 −TagRFP and REC8 −mRUBY 3
progresses until the end of prophase I where the chromosomes are fully condensed. Simultaneously,
MBD6 −GFP expression increases as well, visualized by the extension of the threads
along the chromosomes as well as areal accumulation of CG methylation near the nucleolus
(Fig. 13 - Fig 15). Figure 14 shows the lack of colocalization during mid-prophase where
MBD6 −GFP forms clear strands that do not overlap with ASY3 −TagRFP (see white arrows). Strikingly,
MBD6 −GFP partially colocalizes with ASY3 −TagRFP by the middle to end of prophase
(Fig. 15, Fig. 11 right).
32

Figure 12: MBD6 −GFP xASY3 −TagRFP or REC8 −mRUBY 3 during early to mid-prophase.
MBD6 −GFP shows increasing CG methylation. The increase is characterized by extension
of existing spots and threads as well as appearance of new spots (A: left, B: left). Early to
mid-prophase is characterized by strengthening of ASY3 −TagRFP and REC8 −mRUBY 3 signals,
forming thicker strands (A: middle, B: middle). The merged image shows a yellow signal from
the co-localizing MBD6 −GFP and REC8 −mRUBY 3 proteins (B: right)
Figure 13: MBD6 −GFP xREC8 −mRUBY 3 during mid-prophase. MBD6 −GFP shows extending
spots of methylation (A). The stage can be defined by clear strands of chromosomes, visualized
by REC8 −mRUBY 3 (B). Colocalization between MBD6 −GFP and REC8 −mRUBY 3 is displayed by
a yellow signal in the merged picture (C).
33

Figure 14: MBD6 −GFP xASY3 −TagRFP during mid-prophase. MBD6 −GFP visualizes methylation
spreading along chromosomes throughout the meiocyte. Areas of accumulated CG
methylation can be noticed near nucleoli (I-K red arrows). Chromosomes are illustrated by
aligning ASY3 −TagRFP , which does not colocalize with MBD6 −GFP (H, K white arrows).
34

Figure 15: MBD6 −GFP x ASY3 −TagRFP during middle to late prophase The chromosomes are
in a highly condensed form (B) and heavily methylated (A). MBD6 −GFP and ASY3 −TagRFP
colocalize, displayed by a yellow signal in the merged picture (C).
Figure 16: MBD6 −GFP xASY3 −TagRFP during Prophase I to Metaphase I transition. At the
end of prophase I the chromosomes are higly condensed and methylated (A-C). They then align
in the equatorial plane in metaphase I (D-N).
At the transition to metaphase I, the chromosomes are highly condensed and visible as methylated
dots starting to align to the equatorial plane of the cell. As the synaptonemal complex disappears
in mid to end of prophase, ASY3 −TagRFP is visible as small dots, reflecting protein degradation
(Fig. 16).
Further proceeding to anaphase I, the chromosomes remain visible. The homologous chromosomes
are separated and segregate towards the opposite poles of the cell (Fig. 17 C-H).
The transition into telophase I is presented by the reappearing of the diffuse circular signal of
35

MBD6 −GFP , indicating the reappearance of the nucleus and the nuclear envelope (Fig. 17 I-L).
The second meiotic division continues with prophase II, metaphase II, anaphase II and telophase
II, following the same dynamics as the first meiotic division with the main difference being the
separation of sister chromatids instead of homologous chromosomes (Fig. 18, Fig. 19).
Figure 17: MBD6 −GFP during anaphase I to telophase I transition. MBD6 −GFP follows the
chromosome movement in anaphase II (C-H) as well as in telophase II (I-L).
Figure 18: MBD6 −GFP during prophase II to metaphase II transition. The disappearing
nuclear envelope, displayed by the loss circular MBD6 −GFP signal, indicates the end of prophase
II (B-C). MBD6 −GFP stays attached to the chromosomes during their alignment in the equatorial
plane (D-N.)
36

Figure 19: MBD6 −GFP during metaphase II to telophase II transition. The chromosomes
remain CG methylated throughout meiosis II. MBD6 −GFP follows the alignment of the chromosomes
in the equatorial plane (A-D), the separation of the sister chromatids and their movement
to the opposite end of the cell (E-I) as well as theit reorganization in telophase II (J-P).
In conclusion, CG methylation, visualized with MBD6 −GFP , is present at the chromosomes
throughout meiosis. The methylation is low during pre-meiosis and represented by a dot-like pattern.
When the cell progresses into prophase I, MBD6 −GFP aligns along the chromosome strands.
Additionally, it co-localizes with REC8 −mRUBY 3 in mid-prophase and with ASY3 −TagRFP by the
middle to end of prophase. Further on, the MBD6 −GFP signal remains strong until the end of
meiosis at telophase II.
In addition, a preliminary analysis of the duration of meiotic substages was done. The obtained
data are presented in the Supplementaries.
37

5.2 Detection of CHH methylation with SUVH9 −GFP
To detect asymmetrical CHH methylation, SUVH9 −GFP was used as reporter protein coupled
with ASY3 −TagRFP as a meiotic reporter. Another variant of the double construct using
REC8 −mRUBY 3 did not work as no signal was obtained from any plant. As SUVH9 −GFP did not
show any specific localization at potential centromeric regions, it was not used in combination
with TagRFP− CENH3 due to a lack of achievable informative content.
As described previously, pre-meiosis was defined by central localization of the nucleolus and
squared cell shape (Fig. 20). During this stage, SUVH9 −GFP gives a global signal for asymmetrical
CHH methylation with a lower intensity in the meiocytes compared to the surrounding
tapetum cells (Fig. 20).
Figure 20: SUVH9 −GFP during pre-meiosis. CHH methylation is visualized by SUVH9 −GFP
(A). The pre-meiotic stage can be defined by the central positioning of the nucleolus (C), as well
as by the lack of expression of meiotic ASY3 −TagRFP (not shown).
The expression of ASY3 −TagRFP and the lateral positioning of the nucleolus represent the
transition to early prophase I (Fig. 21). By then, SUVH9 −GFP displays a diffuse distribution
of CHH methylation that increases gradually in intensity towards the end of prophase I (Fig.
21, 22). When the cell reaches metaphase I, a faint diffuse signal appears in the cytoplasm and
persists until the end of telophase II (Fig. 22 C, Fig. 23, Fig. 24). In the nucleus, no distinct
CHH methylation pattern can be observed from metaphase I to telophase I. After telophase I,
SUVH9 −GFP localizes again in the two newly established nuclei (Fig. 23).
38

Figure 21: SUVH9 −GFP during early prophase. ASY3 −TagRFP shows a mainly diffuse signal
with thin theads (B). The nucleolus is in lateral position. Asymmetrical CHH methylation is
shows an unspecific pattern in the nucleus (A).
Further on, SUVH9 −GFP shows similar dynamics in meiosis II, as it gives no distinct nuclear
pattern once the nuclear envelope disappeared. In metaphase II, it is notably not localized in the
organelle band. The signal then reappears in the four nuclei in telophase II (Fig. 24).
As a conclusion, SUVH9 −GFP is highly dynamic during meiosis. It is present in the nucleus
when a nuclear envelope is established thus during pre-meiosis, prophase and telophase. Once
the cell enters metaphase I, a diffuse signal appears in the cytoplasm and remains until telophase
II.
39

Figure 22: SUVH9 −GFP during prophase. A: Early to mid-prophase. The ASY3 −TagRFP
signal increases, the chromosomes are further condensing. SUVH9 −GFP shows a global signal
in the nucleus. B: Mid-prophase. ASY3 −TagRFP keeps further aligning to the condensing
chromosomes. CHH methylation in the nucleus remains global. C: Middle to late prophase.
(left) ASY3 −TagRFP is completely aligned to fully condensed chromosomes. The CHH pattern
remains unchanged. (right) Metaphase I. The SUVH9 −GFP signal in the nucleus disappeared
and shows faint fluorescence in the cytoplasm.
40

Figure 23: SUVH9 −GFP during meiosis I. The beginning of metaphase I is characterized by
the reduction of ASY3 −GFP to red dots (C). SUVH9 −GFP gives a global signal in the nucleus
during interkinesis and prophase II and then disappears in metaphase II (B-F). An observable
signal of SUVH9 −GFP in the nuclei is present in telophase II (G-I).
Figure 24: SUVH9 −GFP during meiosis II. In prophase II SUVH9 −GFP displays a diffuse CHH
methylation pattern in the nucleus as well as a lower level signal in the cytoplasm(A-B). The
nucleolar signal disappears in metaphase II. A specific region, free of SUVH9 −GFP is observed
in the middle of the two chromosome pools (C-I). After nuclear envelope formation in telophase
II within the daughter cells, SUVH9 −GFP localizes within these nuclei, shown by a bright signal
(K-T).
41

6 Discussion
6.1 Comparison of MBD6 −GFP and SUVH9 −GFP dynamics
MBD6 −GFP and SUVH9 −GFP were developed to specifically bind to CG and CHH methylation,
respectively. Thus, a difference in binding patterns was expected as both mainly bind to
heterochromatic regions but CG is more widespread than CHH methylation. Furthermore, gene
body methylation is almost exclusively at CG sites [To et al., 2015].
The binding behaviours of MBD6 −GFP and SUVH9 −GFP are significantly different. Generally,
MBD6 −GFP aligns to chromosomes with a low background signal (Fig. 14). In contrast,
SUVH9 −GFP gives a more global signal (Fig. 22).
During pre-meiosis, they both show low levels of methylation, although MBD6 −GFP shows
additional spots of accumulated CG methylation that are missing with SUVH9 −GFP (Fig. 7, Fig.
20). From these observations it can be concluded that meiocytes are generally low methylated
during pre-meiosis, lacking CG and CHH methylation compared to somatic cells.
Another similarity is that methylation in both contexts increases throughout prophase I, indicated
by increased signals of their respective reporters. The gain of the signal is displayed differently
by the two reporters. While MBD6 −GFP starts forming strands by aligning to the chromosomes
(Fig. 15), SUVH9 −GFP increases in intensity without any change in the global distribution (Fig.
22).
A difference between the two reporters is observed when the cell enters metaphase I. MBD6 −GFP
sticks to the chromosomes following their alignment to the cell equatorial plane in metaphase I as
well as during their segregation in anaphase I. The nuclear reorganization in telophase throughout
meiosis I and meiosis II does not affect this methylation pattern (Fig. 16 - Fig. 19). SUVH9 −GFP
on the other hand disappears from the nucleus as soon as the cell proceeds to metaphase I/II and
is found in the cytoplasm instead. Furthermore, the signal of SUVH9 −GFP reappears when the
nucleus is reestablished in telophase I/II (Fig. 23, Fig. 24). Thus, no chromosome dynamics can
be observed with SUVH9 −GFP .
In summary, both reporters reflect several changes of methylation levels from pre-meiosis to
the end of prophase I. MBD6 −GFP is a good chromosome marker and can be used as a meiotic
hallmark especially during prophase-metaphase-anaphase transitions. It furthermore gives only
a low background signal indicating high binding activity. SUVH9 −GFP can hardly be used to
follow chromosome dynamics as it gives a rather diffuse signal and vanishes from the nucleus
from metaphase I/II to telophase I/II.
42

6.2 Dynamics of MBD6 −GFP
The dynamics of CG methylation were observable due to the good binding activity of MBD6 −GFP
to the chromosomes throughout meiosis. This allowed monitoring of chromosome dynamics as
well.
CG methylation is low during pre-meiosis
Although no pre-meiotic marker was used, pre-meiosis was defined by central localization
of the nucleolus, squared cell shape, a diffuse distribution of REC8 −mRUBY 3 and the lack of
ASY3 −TagRFP expression. Compared to the surrounding somatic cells, the level of CG methylation
in the meiocytes is low (Fig. 7, Fig. 8). Single clear dots, potentially centromeric or
telomeric regions, localized close to the nuclear envelope or nucleolus during pre-meiosis.
The overall low methylation can have various reasons. Since the DNA undergoes replication prior
to meiosis, the low signal of MBD6 −GFP may indicate a hemimethylated state of the DNA produced
by newly synthesized and thus unmethylated DNA strands. This hemimethylation would be
undetectable for MBD6 −GFP . Furthermore, it is known from literature that transposable elements
are activated during gamete development. These transposons that so far escaped the silencing by
the RdDM pathway can now get activated. The reactivation ensures the production of siRNAs that
were already silenced by RdDM pathway and thus reinforces transposon silencing. This process
allegedly takes place during gametogenesis [Law and Jacobsen, 2010]. Nevertheless, it may take
place during sporogenesis as well, as usually silenced transposable elements are transcribed during
prophase [Gutierrez-Marcos and Dickinson, 2012]. This seems reasonable since the genome
is already in a demethylated state after replication and this favorable environment allows the
activation of the transposons. This is further supported by the general loss of heterochromatin in
the spore mother cell, induced by epigenetic reprogramming and the change of genetic program.
Although it was suggested that transposons may be kept silent by other pathways than heterochromatin
formation, no specific mechanisms were identified yet. Meiotic genes are activated in the
SMC during mitosis-meiosis transition, a process that is controlled by epigenetic modifications.
To rebuild pluripotency for the later gametophyte, epigenetic marks of the somatic program need
to be removed [She and Baroux, 2014, Gutierrez-Marcos and Dickinson, 2012].
By using MBD6 −GFP we observed that the CG methylation is low during pre-meiosis. This
demethylation can occur passively or actively. It may serve a distinct purpose like transposon
silencing and epigenetic reprogramming but it also can be a residue of previous DNA
43

eplication.
CG methylation at pericentromeric heterochromatin
MBD6 −GFP partially colocalizes with TagRFP− CENH3 (Fig. 9). Since TagRFP− CENH3 displays
the localization of the centromeres, the dots of CG methylation observed during pre-meiosis may
display centromeric and pericentromeric heterochromatin. The lack of the full overlap may imply
CG hypomethylation of CENH3-binding chromatin domains as reported by [Zhang et al., 2008].
On the other hand, the missing full overlap can simply indicate a steric hindrance of the reporters,
making it impossible for TagRFP− CENH3 and MBD6 −GFP to bind at the same regions. Thus, no
valid statement can be made about the methylation level at the centromere with these reporters.
Furthermore, regions of CG methylation that do not associate with centromeres can be observed.
These dots may display other heterochromatic regions of the genome like telomeres.
Moreover, the spots of CG methylation seem to always localize at the nuclear envelope or
nucleolus (Fig. 7, Fig. 8). The association with the nuclear envelope fits to previous observations
of centromeres being attached to the nuclear periphery during mitotic interphase and may suggest
similar mechanisms in pre-meiosis. The impression of centromeres being at the nucleolus may
be a side effect of this specific point of view and spatial positioning of the nucleus as the images
are only displayed in a 2D view. [Fang and Spector, 2005]. Thus it can be assumed that all of
the centromeres localize at the nuclear envelope.
CG methylation during meiosis
While the CG methylation was low during pre-meiosis, it accumulates throughout prophase I.
The increase is small in early prophase I, shown by slightly extending methylation along the
chromosomes. But at the end of prophase I, they are highly methylated as the methylation
stretches along most chromosome regions. This impression of high methylation can either
originate from the very condensed form of the chromosomes at this point of time without a real
increase in methylation or from an active gain of methylation. Active methylation may have
the purpose to reestablish symmetric CG methylation previously lost due to DNA replication or
silence transposons that were activated in pre-meiosis. A combination of active remethylation
and high chromosome condensation is a possible explanation as well. Throughout meiosis, the
chromosomes remain in their highly condensed and methylated state.
Not only the methylation increases with ongoing prophase I, but also the alignment of REC8 −mRUBY 3
and ASY3 −TagRFP reveals a dynamic pattern. Towards the end of prophase I, they are fully
44

aligned to the chromosomes, visualized as thick strands. As both of them extent along the chromosomes,
REC8 −mRUBY 3 partially colocalizes with MBD6 −GFP in early to mid-prophase while
ASY3 −TagRFP does not until the middle to end of prophase (Fig. 12 and 15). In mid-prophase,
it is clearly observable, that ASY3 −TagRFP does not align in methylated regions in contrast
to REC8 −mRUBY 3 (Fig. 13 and 14 white arrow). ASY3 mainly plays a role in crossing-over
induction and the formation of the synaptonemal complex. In regions where ASY3 is aligned,
recombination is possible or the other way around, ASY3 aligns to regions where recombination
is available. It can be possible that ASY3 does not align to heavily methylated regions, as
they might be recombination-suppressed regions. Furthermore, the timing of recombination
initiation plays an important role in crossing-over formation. Regions with early initiation of
recombination show a preference in crossing-over formation [Lambing et al., 2017]. This may
explain why methylated region are excluded from ASY3 −TagRFP alignment until late prophase I
as they are not supposed to recombine. Moreover, methylation may also suppress ASY3 −TagRFP
alignment for the same reasons. REC8 −mRUBY 3 on the other hand seems to be less influenced by
CG methylation than ASY3 −TagRFP . This is probably due to its primary role in sister chromatid
cohesion, a function that should be less impaired by methylation as methylated regions have
to keep their cohesion as well. Nevertheless, it has to be noted that the lack of colocalization
can be a consequence of steric hindrance between MBD6 −GFP and ASY3 −TagRFP . It can not
be observed whether the two reporter proteins influence each other in their binding activity.
Clearly visible is also the signal of MBD6 −GFP at the nucleolus (Fig. 10 - Fig. 15). Around the
nucleolus, nucleolar organizer regions (NORs) accumulate. They contain the rRNA genes for
ribosome biosynthesis. rRNA genes are widely variable and their expression largely depends on
the current needs of the cell. On average, about half of the rRNA genes are active. Inactive rRNA
genes are DNA methylated at CG contexts. The spatial distribution of the NORs with rRNA
genes depends on the active or inactive state of the chromatin. Active rRNA genes are localized
within the nucleolus while inactive rRNA genes are excluded, resulting in the localization of the
respective NORs outside of the nucleolus (Fig. 25). Although all active rRNA genes are within
the nucleolus, not all genes that are within the nucleolus are active. About 20 % of the genes
are inactive and heavily methylated. Presumably, they are moved to the nucleolus due to the
close neighborhood with active genes [Pontvianne et al., 2013]. Thus, the MBD6 −GFP signal in
the nucleolus most likely reflects these heavily methylated rRNA genes. The signal around the
nucleolus may originiate from the NORs of inactive and highly CG methylated rRNA genes that
accumulate externally. This external accumulation can also explain the color difference between
nucleolar and nucleus methylation.
45

Figure 25: Localization of rRNA genes. A: Active, thus unmethylated rRNA genes are localized
inside the nucleolus. Methylated, silenced rRNA genes locate in the nucleoplasm. B: Silenced
rRNA genes form condensed knobs outside the nucleolus while active genes are transcribed
inside the nucleolus. Figure from [Pontvianne et al., 2013]
Binding activity of MBD6 −GFP
MBD6 −GFP has a high binding activity. It is constantly bound to chromosomes and consequently
gives low to no background signal. Thus it is a good chromosome reporter line.
Ingouff et al., (2017) estimated the binding specificity of MBD6 −GFP with various tests. While
binding to CHH methylation was disproved, binding to CHG methylation was not ruled out.
Furthermore, comparison of MBD6 −GFP ChIP data with MeDIP data set indicates a high
correlation. But since MeDIP data recognize methylation in all contexts, this is not sufficient to
prove that MBD6 −GFP only binds methylated CG, even if CG is the most commonly methylated
context in A. thaliana. Hence, it can not be concluded that MBD6 −GFP exclusively binds to CG
methylation.
MBD6 −GFP detects methylation but cannot be used for quantitative measurements. It does enrich
at heavily methylated transposable elements but also at low methylated gene bodies. Therefore,
MBD6 −GFP cannot be used to make definitive statements of quantitative methylation levels at
certain regions. Still it can be used to observe overall changes in methylation levels.
6.3 Dynamics of SUVH9 −GFP
SUVH9 −GFP gives a diffuse global signal in the nucleus for CHH methylation in pre-meiosis,
prophase and telophase.
46

Low signal of SUVH9 −GFP during pre-meiosis
In pre-meiosis, the signal of SUVH9 −GFP is low compared to the surrounding somatic cells,
indicating a low CHH methylation (Fig. 20). Since CG methylation was reduced as well
during this phase, a general reduction of methylation is hinted. CHH methylation is mainly
used in heterochromatin formation, thus playing a role in transposon silencing. The low signal
in pre-meiosis may be due to the above mentioned derepression of transposons to reestablish
silencing by the RdDM pathway with the purpose to "catch" previously active transposons.
This reassurance pathway of transposon silencing fits to the general knowledge about the great
importance of transposable element inactivation in plants. Allegedly, non-CG methylation
developed for that purpose [Kato et al., 2003]. The low CHH methylation may also result from
previous DNA replication and the slow remethylation of newly replicated strands which in case
of CHH methylation, equals de novo methylation. Little is known about how fast methylation
of newly replicated DNA strands occurs after replication. Since a demethylated state due
to replication would promote transposon activation, a combination of both is also possible.
As CHH methylation is probably also involved in gene expression control and non-heritable
stress responses, epigenetic reprogramming may contribute to the lack of DNA methylation as
well.
SUVH9 −GFP during meiosis
Throughout prophase I, the signal of SUVH9 −GFP increases in intensity, hinting an increase
of CHH methylation (Fig. 22). This gain in CHH methylation may be explained by either
remethylation after DNA replication or silencing of pre-meiotically active transposons.
When reaching metaphase I/II, the signal of SUVH9 −GFP quickly disappears within the nucleus.
Instead, it can be observed in the cytoplasm (Fig. 23). Either demethylation or release of the
protein from the DNA can explain this change. Demethylation would indicate a highly dynamic
behavior of CHH methylation throughout meiosis, as the vanishing can be observed both during
metaphase I and metaphase II with an instant remethylation in interkinesis/prophase II and
telophase II, respectively. Alternatively, the loss of signal in the nucleus may be due to the
release of the protein instead of demethylation. The SUVH9 −GFP protein may be transferred
to the cytoplasm to not disrupt the chromosome separation. Clearly visible, the protein is not
localized in the organelle band that forms between the dividing cells but in the cytoplasm of
the respective cells (Fig. 24). Within the cytoplasm, SUVH9 −GFP may be degraded and thus
displays a lower signal. Another possibility is that the protein is protected from degradation by
47

storage in vesicles to be then re-used in telophase. Compared to the nucleus, the lower signal
intensity observed in the cytoplasm may then be due to the greater spatial distribution of the
protein-containing vesicles within the cytoplasm.
Binding activity of SUVH9 −GFP
It is difficult to understand the global-wide localization of SUVH9 −GFP in the nucleus. A
diffuse distribution may indicate that the reporter protein is not binding properly to the DNA, the
localization of the protein in nucleus however disproves this as unbound protein would localize
in the cytoplasm. The disappearance of SUVH9 −GFP from the nucleus in metaphase I/II is most
likely due to protein release from the chromosomes and following degradation or protection in
vesicles.
In summary, SUVH9 −GFP can be a useful reporter to measure CHH methylation level altough its
global distribution and protein dynamics cannot be fully explained. All interpretations have to be
taken with caution but still may give useful informations about the behavior of CHH methylation
during meiosis for future studies.
6.4 General remarks on the use of fluorescent proteins
Double constructs are a useful instrument for observation of the dynamics and interplay of two
proteins. In this study, they were used to stage the cells with a known meiotic reporter while the
focus was on the observation of the methylation reporters with unknown dynamics. Still, the
functionality of these double constructs is not guarantied. The usual procedure of testing the
functionality of the constructs by introducing them into their respective mutant plants cannot
be applied. Producing double mutants for both proteins is often lethal for the plants. Thus a
negative influence of the reporter proteins to each other cannot be ruled out. Functionality of a
transgene within the plant does not guarantee its workability as part of a double construct.
An additional point that has to be considered is that the constructs were introduced into wildtype
plants which also express their endogenous protein variant. Furthermore, the transgenes were
combined with enhancing promoters to induce an increased expression. Combining these two
factors it is clear that the quantity of the protein within the plant is above physiological conditions.
Since protein levels are usually strictly controlled by multiple pathways within each cell, this
may lead to changes in the protein expression.
48

Another problem lies within the nature of fluorescent proteins. While using them, it has to be
assumed that they behave like an unmodified protein. Nevertheless, there is the possibility of the
protein behaving differently due to the tag. The binding specificity and interaction ability can be
influenced. This is escpecially the case for fluorophores that are expressed under the control of
an enhancing promotor [Crivat and Taraska, 2012]. Thus, observations made with fluorescence
proteins should be backed up with other study methods.
6.5 Outlook
Additional studies will be needed to confirm the findings and further explore the methylation
dynamics. For this purpose, different mutants can be used to observe whether changes occur.
Mutants with meiosis defects would be suitable like rbr1-2, dyad or the CDKA;1 mutants DE
and DB-dead to check for changes in the methylation dynamics connected to meiotic disruptions.
Plants that are defective in methylation establishment may be interesting as well, MET1 and
DDM1 would be good candidates. Furthermore, monitoring of the female meiosis with these
reporters and mutant lines could be done. Currently this is not possible due to a lack of methods.
Generally, it may be useful to further explore the binding activity and specificity of both reporters.
All findings made within this study should be backed up with other study methods such as
expression analysis. For example, the observed demethylation with subsequent remethylation
could be supported by expression analysis of respective demethylases ROS1, DML2 and DML3
and methyltransferase MET1 or activity of the RdDM pathway.
6.6 Conclusion
In summary, it can be stated that CG and presumably also CHH methylation are highly dynamic
throughout pre-meiosis and meiosis. In pre-meiosis, the chromosomes are CG methylated at
pericentromeric heterochromatin and few centromere-independent heterochromatic regions. By
the beginning of the prophase, the methylation level increases until the chromosomes reach
a highly methylated state at the end of prophase. CG methylation may have an influence on
alignment of recombination-associated proteins such as ASY3, as it does not align to methylated
regions until mid to late prophase. The chromosomes remain heavily CG methylated until the
end of meiosis. Thus, MBD6 −GFP is a useful reporter to follow chromosome movements and
methylation dynamics, although a context-related binding unspecificity cannot be eliminated.
On the contrary, SUVH9 −GFP turned out to be useful for CHH methylation observations but not
49

for chromosome movements since the signal was diffuse throughout monitoring. Further studies
will be required to confirm the findings.
50

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56

7 Eidesstaatliche Erklärung
Hiermit erkläre ich an Eides statt, dass die vorliegende Arbeit von mir selbständig verfasst
wurde und ich keine anderen als die angegebenen Hilfsmittel – insbesondere keine im Quellenverzeichnis
nicht benannten Internetquellen – benutzt habe und die Arbeit von mir vorher
nicht einem anderen Prüfungsverfahren eingereicht wurde. Die eingereichte schriftliche Fassung
entspricht der auf dem elektronischen Speichermedium. Ich bin damit einverstanden, dass die
Bachelorarbeit veröffentlicht wird.
57

8 Supplementaries
8.1 Movies
MBD6 −GFP xASY3 −TagRFP

MBD6 −GFP xREC8 −TagRFP

SUVH9 −GFP xASY3 −TagRFP

8.2 Additional graphics
Figure 26: MBD6 −GFP xREC8 −mRUBY 3 during meiosis I Disappearance of the nuclear envelope
represents the end of prophase I (A-C). The chromosomes align in the equatorial plane during
metaphase (D-F) and are segregated in anaphase I (G-H). The nuclear envelope reappears in
telophase I, represented by the diffuse circular signal of MBD6 −GFP (I).

Figure 27: MBD6 −GFP xREC8 −mRUBY 3 during meiosis II The transition to metaphase II is
represented by the diappearing nuclear envelope (A-D). The chromosome alignment in the
equatorial plane(E) to be then seperated in anaphase II (F-G). In telophase II, the nuclear
envelope reappears, visualized by the circular signal of MBD6 −GFP (H-L).
8.3 Duration of meiotic substages
By comparing the duration of the different meiotic substages, differences between the anthers can
be observed. Within the anthers themself, the meiocytes progress synchronized through meiosis
with just minor deviations. All meiocytes spent the most time during telophase I/prophase II.
Furthermore, the greatest difference in duration can be noted at this stage, as anthers 1 and 2
spend 128 minutes and 125 minutes at this stage respectively, while anther 3 remains 55 minutes
and anther 4 took 40 minutes until further progression. It has to be noted that anther 1 and
2 originate from the same plant and were observed simultaneously. On the contrary, no such
deviation can be noted within the other phases. Metaphase I and II are of similar lengths in
all meiocytes, between 25 minutes and 35 minutes. Moreover, anaphase I and II show similar
durations as well, both ranging between 10 minutes and 20 minutes in all meiocytes (Fig. 28.)
Thus, the overall time course from entrance to metaphase I to telophase II ranges between 120
min and 222 min. These observations fit to values from the literature, that report completion of
meiosis within 180 mins once metaphase I is reached [Armstrong et al., 2003]. The deviation
from this value of up to 42 mins, shown by anthers 1 and 2, may be due to differences in room
temperature at the time of observation. This distribution of substage lengths is as expected
and reasonable, considering the processes that take place during the respective phases. In
telophase I/prophase II nuclear reorganization takes place including chromatin decondensation
and subsequent condensation to proceed to metaphase. During metaphase on the other hand,

the chromosomes need to organize in the equatorial plane, which may take more time then the
following separation in anaphase.
Figure 28: Lengths of meiotic stages. Duration of meiotic substages devided by observed
anthers. Anther 1 and anther 2 consisted of 13 meiocytes each, anther 3 inherited 6 meiocytes
and within anther 4 3 meiocytes were observed. The progression of meiosis within the anthers
was synchronized with deviations of only a few minutes. These deviations are negligible.
8.4 Vector Sequences and Maps
SUVH9 −GFP
pENTR2B-pHTR5-SUVH9-NLS-GFP-RBCS

CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT ACCGCTAGCA TGGATC-
TCGG GGACGTCTAA CTACTAAGCG AGAGTAGGGA ACTGCCAGGC ATCAAATAAA
ACGAAAGGCT CAGTCGGAAG ACTGGGCCTT TCGTTTTATC TGTTGTTTGT CGGT-
GAACGC TCTCCTGAGT AGGACAAATC CGCCGGGAGC GGATTTGAAC GTTGTGAAGC
AACGGCCCGG AGGGTGGCGG GCAGGACGCC CGCCATAAAC TGCCAGGCAT CAAAC-
TAAGC AGAAGGCCAT CCTGACGGAT GGCCTTTTTG CGTTTCTACA AACTCTTCCT
GTTAGTTAGT TACTTAAGCT CGGGCCCCAA ATAATGATTT TATTTTGACT GATAGT-
GACC TGTTCGTTGC AACAAATTGA TAAGCAATGC TTTTTTATAA TGCCAACTTT
GTACAAAAAA GCAGGCTGGC GCCGGAACCA ATTCAGTCGA CTGGATCCtc cggatccaga
tccgatataa caaaatttga atcgcacaga tcgatctctt tggagattct atacctagaa aatggagacg attttcaaat ctctgtaaaa
attctggttt cttcttgacg gaagaagacg acgactccaa tatttcggtt agtactgaac cggaaagtttgactggtgca accaatttaa
tgtaccgtac gtaacgcacc aatcggattt tgtattcaat gggccttatc tgtgagccca ttaattgatg tgacggccta aactaaatcc
gaacggttta tttcagcgat ccgcgacggt ttgtattcag ccaatagcaa tcaattatgt agcagtggtg atcctcgtca aaccagtaaa
gctagatctg gaccgttgaa ttggtgcaag aaagcacatg ttgtgatatt tttacccgta cgattagaaa acttgagaaa cacattgata
atcgataaaa accgtccgat catataaatc cgctttacca tcgttgccca taaattaata tcaatagccg tacacgcgtg aagactgaca
atattatctt tttcgaattc ggagctcaag tttgaaattc ggagaagcta gagagttttc tgaggtacga ttcttcgatc ctctttgatt
ttcctggaaa tattttttcg gtgatcgtga aactactgga atcgctcgat aggtggtacg aaattaggcgagattagttt ctattcttgg
ccattatctt gtttcttcgc cgaatgatct tccggataaa gattttaggt tagagatgaa tcgtatagct agatttcatc accagatagt
ttctttgtct agaatctctg aaattctcga tagttttcac atgtgtaaat agattgttct tattcggcga ttgttgatta gggttttgat
tttcttgatt atgcgattgc aattagggat tttctttggt tttgtgttga tcttacgatacattccggca attgaatacg tatggatcta
aatcttgtta atttgttgaa cagggtacca acaGTGGATC Catgggttct tctcacattc ctcttgatcc gtctctaaat ccgtcacctt
cactgatccc aaagcttgaa ccagtcactg aatcaaccca aaacttggcc tttcaacttc caaacacaaa cccacaagcc

ctaatttcat cagccgtctc cgatttcaac gaagccacag acttttcctc agattacaac accgtcgccg agtcagcccg gtctgctttc
gctcaacggc ttcaacgtca cgatgatgtt gcggttcttg attccttaac cggagcaatc gtaccggttg aggagaatcc ggaaccggaa
ccgaatcctt actcaaccag tgactcttca ccgtcggttg ctactcaacg acctagaccg cagccacgtt cgtcggagct
agtgaggatc actgatgttg gacctgaaag tgagagacag tttcgtgaac atgtgaggaa gacgagaatg atttatgatt ctcttaggat
gtttttgatg atggaagaag ctaaacgtaa tggggttggt ggaagaagag ctagagctga tggtaaagct ggtaaagctg
gttcaatgat gagagattgt atgttgtgga tgaatcgtga taaacgaatc gtcggttcga ttcccggtgt tcaagttggg gatatcttct
tctttaggtt tgagttatgt gtcatgggct tacatggaca ccctcaatct gggatcgatt ttcttacagg gagtcttagt tctaatgggg
agccaatagc tactagtgtg attgtttctg gtgggtatga ggatgatgat gatcaaggag atgtgattat gtatacaggt cagggtggac
aagataggct tggaaggcaa gctgaacatc agaggctgga aggtggaaac cttgcgatgg aacggagtat gtattatggg
attgaagtga gagttattag agggttgaag tatgagaatg aggtttctag tagagtttat gtttatgatg ggttgtttag gattgttgat
tcttggtttg atgttgggaa gtctggtttc ggtgtgttta aatatcggttggagaggatt gaaggacagg ctgagatggg tagttcggtg
ttgaagtttg ctaggactcttaagactaat ccattgtctg tgaggccgag aggttacatc aatttcgata tctcgaatgg gaaggagaat
gttcctgtct atttgtttaa cgacattgac agcgatcaag aacctttgtattatgagtat cttgcgcaaa cttcgtttcc tcctggctta
tttgttcagc aaagtggtaatgcaagtgga tgtgactgtg tcaatggttg tggcagtggc tgcctttgtg aagccaagaa ttcaggtgag
attgcttatg attataatgg gacgctaata agacagaaac cgttgataca tgaatgtgga tcagcgtgtc agtgccctcc aagctgtcga
aaccgtgtga ctcaaaaggg tttgaggaat aggctagaag tgtttaggtc actggaaacg ggttggggag ttcggtcttt
ggatgtatta catgctggtg cttttatttg tgagtatgct ggggttgctt tgacaagggaacaagccaac attctgacca tgaacggtga
tacattggta tatcctgctc gtttctcttctgcgagatgg gaagattggg gagatctgtc tcaggtcctt gctgatttcg agcggccttc
ttatcctgat attcctccgg ttgatttcgc aatggatgtg tccaagatga ggaatgttgc ttgttacata agccacagta ctgatccgaa
tgtcattgtc cagtttgtac tccatgatca caacagtctc atgttcccta gagtcatgct tttcgctgct gaaaacatcc ctcccatgac
tgagctcagc cttgattacg gagtagttga tgattggaac gccaagcttg ccatttgtaa tggcggcggc ggccctaaga agaagagaaa
ggttggTACC GGctccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt cacctacggc gtgcagtgct tcagccgcta ccccgaccac
atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcacccagtccaagctg agcaaagacc ccaacgagaa
gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc acggcatgga cgagctgtac aagtcaggtg
gatcctagat aacctttacc ttcattccct gtaaattggt accctgcaaa gctttcgttc gtatcatcgg tttcgacaac gttcgtcaag
ttcaatgcat cagtttcatt gcgcacacac cagaatccta ctgagtttga gtattatggc attgggaaaa ctgtttttct tgtaccattt
gttgtgcttg taatttactg tgttttttat tcggttttcg ctatcgaact gtgaaatgga aatggatgga gaagagttaatgaatgatat
ggtccttttg ttcattctca aattaatatt atttgttttttctcttattt gttgtgtgtt gaatttgaaa ttataagaga tatgcaaaca ttttgttttg

agtaaaaatg tgtcaaatcg tggcctctaa tgaccgaagt taatatgagg agtaaaacac ttgtagttgt accattatgc ttattcacta
ggcaacaaat atattttcag acctagaaaa gctgcaaatg ttactgaata caagtatgtc ctcttgtgtt ttagacattt atgaactttc
ctttatgtaa ttttccagaa tccttgtcag attctaatca ttgctttata attatagtta tactcatgga tttgtagttg agtatgaaaa tattttttaa
tgcattttatgacttgccaa ttgattgaca acatgcatca atcgGCGGCC GCACTCGAGA TATCTAGACC
CAGCTTTCTT GTACAAAGTT GGCATTATAA GAAAGCATTG CTTATCAATT TGTTG-
CAACG AACAGGTCAC TATCAGTCAA AATAAAATCA TTATTTGCCA TCCAGCTGCA
GCTCTGGCCC GTGTCTCAAA ATCTCTGATG TTACATTGCA CAAGATAAAA ATATAT-
CATC ATGAACAATA AAACTGTCTG CTTACATAAA CAGTAATACA AGGGGTGTTA
TGAGCCATAT TCAACGGGAA ACGTCGAGGC CGCGATTAAA TTCCAACATG GAT-
GCTGATT TATATGGGTA TAAATGG GCT CGCGATAATG TCGGGCAATC AGGTGC-
GACA ATCTATCGCT TGTATGGGAA GCCCGATGCG CCAGAGTTGT TTCTGAAACA
TGGCAAAGGT AGCGTTGCCA ATGATGTTAC AGATGAGATG GTCAGACTAA ACTG-
GCTGAC GGAATTTATG CCTCTTCCGA CCATCAAGCA TTTTATCCGT ACTCCTGATG
ATGCATGGTT ACTCACCACT GCGATCCCCG GAAAAACAGC ATTCCAGGTA TTAGAA-
GAAT ATCCTGATTC AGGTGAAAAT ATTGTTGATG CGCTGGCAGT GTTCCTGCGC CG-
GTTGCATT CGATTCCTGT TTGTAATTGT CCTTTTAACA GCGATCGCGT ATTTCGTCTC
GCTCAGGCGC AATCACGAAT GAATAACGGT TTGGTTGATG CGAGTGATTT TGAT-
GACGAG CGTAATGGCT GGCCTGTTGA ACAAGTCTGG AAAGAAATGC ATAAACTTTT
GCCATTCTCA CCGGATTCAG TCGTCACTCA TGGTGATTTC TCACTTGATA ACCT-
TATTTT TGACGAGGGG AAATTAATAG GTTGTATTGA TGTTGGACGA GTCGGAATCG
CAGACCGATA CCAGGATCTT GCCATCCTAT GGAACTGCCT CGGTGAGTTT TCTC-
CTTCAT TACAGAAACG GCTTTTTCAA AAATATGGTA TTGATAATCC TGATATGAAT
AAATTGCAGT TTCATTTGAT GCTCGATGAG TTTTTCTAAT CAGAATTGGT TAATTG-
GTTG TAACATTATT CAGATTGGGC CCCGTTCCAC TGAGCGTCAG ACCCCGTAGA
AAAGATCAAA GGATCTTCTT GAGATCCTTT TTTTCTGCGC GTAATCTGCT GCTTG-
CAAAC AAAAAAACCA CCGCTACCAG CGGTGGTTTG TTTGCCGGAT CAAGAGC-
TAC CAACTCTTTT TCCGAAGGTA ACTGGCTTCA GCAGAGCGCA GATACCAAAT
ACTGTTCTTC TAGTGTAGCC GTAGTTAGGC CACCACTTCA AGAACTCTGT AGCAC-
CGCCT ACATACCTCG CTCTGCTAAT CCTGTTACCA GTGGCTGCTG CCAGTGGCGA
TAAGTCGTGT CTTACCGGGT TGGACTCAAG ACGATAGTTA CCGGATAAGG CGCAGCG-
GTC GGGCTGAACG GGGGGTTCGT GCACACAGCC CAGCTTGGAG CGAACGACCT
ACACCGAACT GAGATACCTA CAGCGTGAGC TATGAGAAAG CGCCACGCTT CCC-
GAAGGGA GAAAGGCGGA CAGGTATCCG GTAAGCGGCA GGGTCGGAAC AGGA-
GAGCGC ACGAGGGAGC TTCCAGGGGG AAACGCCTGG TATCTTTATA GTCCTGTCGG

GTTTCGCCAC CTCTGACTTG AGCGTCGATT TTTGTGATGC TCGTCAGGGG GGCG-
GAGCCT ATGGAAAAAC GCCAGCAACG CGGCCTTTTT ACGGTTCCTG GCCTTTTGCT
GGCCTTTTGC TCACATGTT
MBD6 −GFP
pENTR2B-pHTR5-MBD-NLS-GFP-RBCS
CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT ACCGCTAGCA TGGATC-
TCGG GGACGTCTAA CTACTAAGCG AGAGTAGGGA ACTGCCAGGC ATCAAATAAA
ACGAAAGGCT CAGTCGGAAG ACTGGGCCTT TCGTTTTATC TGTTGTTTGT CGGT-
GAACGC TCTCCTGAGT AGGACAAATC CGCCGGGAGC GGATTTGAAC GTTGTGAAGC
AACGGCCCGG AGGGTGGCGG GCAGGACGCC CGCCATAAAC TGCCAGGCAT CAAAC-
TAAGC AGAAGGCCAT CCTGACGGAT GGCCTTTTTG CGTTTCTACA AACTCTTCCT
GTTAGTTAGT TACTTAAGCT CGGGCCCCAA ATAATGATTT TATTTTGACT GATAGT-
GACC TGTTCGTTGC AACAAATTGA TAAGCAATGC TTTTTTATAA TGCCAACTTT
GTACAAAAAA GCAGGCTGGC GCCGGAACCA ATTCAGTCGA CTGGATCCtc cggatccaga
tccgatataa caaaatttga atcgcacaga tcgatctctttggagattct atacctagaa aatggagacg attttcaaat ctctgtaaaa
attctggtttcttcttgacg gaagaagacg acgactccaa tatttcggtt agtactgaac cggaaagttt gactggtgca accaatttaa
tgtaccgtac gtaacgcacc aatcggattt tgtattcaatgggccttatc tgtgagccca ttaattgatg tgacggccta aactaaatcc
gaacggtttatttcagcgat ccgcgacggt ttgtattcag ccaatagcaa tcaattatgt agcagtggtgatcctcgtca
aaccagtaaa gctagatctg gaccgttgaa ttggtgcaag aaagcacatgttgtgatatt tttacccgta cgattagaaa acttgagaaa
cacattgata atcgataaaaaccgtccgat catataaatc cgctttacca tcgttgccca taaattaata tcaatagccgtacacgcgtg
aagactgaca atattatctt tttcgaattc ggagctcaag tttgaaattcggagaagcta gagagttttc tgaggtacga ttcttcgatc
ctctttgatt ttcctggaaatattttttcg gtgatcgtga aactactgga atcgctcgat aggtggtacg aaattaggcgagattagttt

ctattcttgg ccattatctt gtttcttcgc cgaatgatct tccggataaagattttaggt tagagatgaa tcgtatagct agatttcatc accagatagt
ttctttgtctagaatctctg aaattctcga tagttttcac atgtgtaaat agattgttct tattcggcgattgttgatta gggttttgat
tttcttgatt atgcgattgc aattagggat tttctttggt tttgtgttga tcttacgata cattccggca attgaatacg tatggatcta
aatcttgttaatttgttgaa cagggtacca acaatgcaaa ctgagtcaaa atctcgaaaa cgggctgctc ccggggacaa ctggttaccg
ccgggctgga gggtcgagga caaaatccgc acatcgggcg caactgcagg atctgttgac aaatattact atgagcccaa
cactggacgt aagttccgaa gccgcacgga agtattatat tatcttgagc agggtacgag caagcgcggg actaaaaaag
cggaaaatac ttacttcaac ccggatcact tcgagggcgg cggcggccag acggaatcga agtcgcggaa gcgtgcggcg
cctggagata attggttgcc tcctggttgg agagttgaag ataagattcg aacttccggt gccacagctg gttcggtgga taagtactat
tacgaaccaa atacaggacg taagtttcgg tccaggactg aggtgctgta ctacttggaa catggaactt ctaaaagagg
caccaagaaa gctgagaata catatttcaa tccagaccat tttgaaggcg gcggcggccc taagaagaag agaaaggttg
gctccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
caagctgccc gtgccctggc ccaccctcgt gaccaccttc acctacggcg tgcagtgctt cagccgctac cccgaccaca
tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagccacaacgtcta tatcatggcc gacaagcaga
agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccaagctga gcaaagaccc
caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactca cggcatggac gagctgtaca
agtcaggtgg atcctagata acctttacct tcattccctg taaattggta ccctgcaaag ctttcgttcg tatcatcggt ttcgacaacg
ttcgtcaagt tcaatgcatc agtttcattg cgcacacacc agaatcctac tgagtttgag tattatggca ttgggaaaac tgtttttctt
gtaccatttg ttgtgcttgt aatttactgt gttttttatt cggttttcgc tatcgaactg tgaaatggaa atggatggag aagagttaat gaatgatatg
gtccttttgt tcattctcaa attaatatta tttgtttttt ctcttatttg ttgtgtgttg aatttgaaat tataagagat atgcaaacat
tttgttttga gtaaaaatgt gtcaaatcgt ggcctctaat gaccgaagtt aatatgagga gtaaaacact tgtagttgta ccattatgct
tattcactag gcaacaaata tattttcaga cctagaaaag ctgcaaatgt tactgaatac aagtatgtcc tcttgtgttt tagacattta
tgaactttcc tttatgtaat tttccagaat ccttgtcaga ttctaatcat tgctttataa ttatagttat actcatggat ttgtagttga gtatgaaaat
attttttaat gcattttatg acttgccaat tgattgacaa catgcatcaa tcgGCGGCCG CACTCGAGAT ATC-
TAGACCC AGCTTTCTTG TACAAAGTTG GCATTATAAG AAAGCATTGC TTATCAATTT
GTTGCAACGA ACAGGTCACT ATCAGTCAAA ATAAAATCAT TATTTGCCAT CCAGCT-
GCAG CTCTGGCCCG TGTCTCAAAA TCTCTGATGT TACATTGCAC AAGATAAAAA
TATATCATCA TGAACAATAA AACTGTCTGC TTACATAAAC AGTAATACAA GGGGT-
GTTAT GAGCCATATT CAACGGGAAA CGTCGAGGCC GCGATTAAAT TCCAACATGG
ATGCTGATTT ATATGGGTAT AAATGGGCTC GCGATAATGT CGGGCAATCA GGTGC-
GACAA TCTATCGCTT GTATGGGAAG CCCGATGCGC CAGAGTTGTT TCTGAAACAT

GGCAAAGGTA GCGTTGCCAA TGATGTTACA GATGAGATGG TCAGACTAAA CTG-
GCTGACG GAATTTATGC CTCTTCCGAC CATCAAGCAT TTTATCCGTA CTCCTGATGA
TGCATGGTTA CTCACCACTG CGATCCCCGG AAAAACAGCA TTCCAGGTAT TAGAA-
GAATA TCCTGATTCA GGTGAAAATA TTGTTGATGC GCTGGCAGTG TTCCTGCGCC
GGTTGCATTC GATTCCTGTT TGTAATTGTC CTTTTAACAG CGATCGCGTA TTTCGTC-
TCG CTCAGGCGCA ATCACGAATGAATAACGGTT TGGTTGATGC GAGTGATTTT GAT-
GACGAGC GTAATGGCTG GCCTGTTGAA CAAGTCTGGA AAGAAATGCA TAAACTT-
TTG CCATTCTCAC CGGATTCAGT CGTCACTCAT GGTGATTTCT CACTTGATAA CCTT-
ATTTTT GACGAGGGGA AATTAATAGG TTGTATTGAT GTTGGACGAG TCGGAATCGC
AGACCGATAC CAGGATCTTG CCATCCTATG GAACTGCCTC GGTGAGTTTT CTC-
CTTCATT ACAGAAACGG CTTTTTCAAA AATATGGTAT TGATAATCCT GATATGAATA
AATTGCAGTT TCATTTGATG CTCGATGAGT TTTTCTAATC AGAATTGGTT AATTG-
GTTGT AACATTATTC AGATTGGGCC CCGTTCCACT GAGCGTCAGA CCCCGTAGAA
AAGATCAAAG GATCTTCTTG AGATCCTTTT TTTCTGCGCG TAATCTGCTG CTTG-
CAAACA AAAAAACCAC CGCTACCAGC GGTGGTTTGT TTGCCGGATC AAGAGC-
TACC AACTCTTTTT CCGAAGGTAA CTGGCTTCAG CAGAGCGCAG ATACCAAATA
CTGTTCTTCT AGTGTAGCCG TAGTTAGGCC ACCACTTCAA GAACTCTGTA GCAC-
CGCCTA CATACCTCGC TCTGCTAATC CTGTTACCAG TGGCTGCTGC CAGTGGCGAT
AAGTCGTGTC TTACCGGGTT GGACTCAAGA CGATAGTTAC CGGATAAGGC GCAGCG-
GTCG GGCTGAACGG GGGGTTCGTG CACACAGCCC AGCTTGGAGC GAACGAC-
CTA CACCGAACTG AGATACCTAC AGCGTGAGCT ATGAGAAAGC GCCACGCTTC
CCGAAGGGAG AAAGGCGGAC AGGTATCCGG TAAGCGGCAG GGTCGGAACA GGA-
GAGCGCA CGAGGGAGCT TCCAGGGGGA AACGCCTGGT ATCTTTATAG TCCTGT-
CGGG TTTCGCCACC TCTGACTTGA GCGTCGATTT TTGTGATGCT CGTCAGGGGG
GCGGAGCCTA TGGAAAAACG CCAGCAACGC GGCCTTTTTA CGGTTCCTGG CCTTT-
TGCTG GCCTTTTGCT CACATGTT
REC8 −mRUBY 3
pENTR-proREC8-REC8-mRUBY3 3’ UTR

CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT ACCGCCTTTG AGTGA-
GCTGA TACC GCTCGC CGCAGCCGAA CGACCGAGCG CAGCGAGTCA GTGAGC-
GAGG AAGCGGAAGA GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG
ATTCATTAAT GCAGCTGGCA CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCG-
CAAC GCAATTAATA CGCGTACCGC TAGCCAGGAA GAGTTTGTAG AAACGCAAAA
AGGCCATCCG TCAGGATGGC CTTCTGCTTA GTTTGATGCC TGGCAGTTTA TGGCGGG
CGT CCTGCCCGCC ACCCTCCGGG CCGTTGCTTC ACAACGTTCA AATCCGCTCC
CGGCGGATTT GTCCTACTCA GGAGAGCGTT CACCGACAAA CAACAGATAA AAC-
GAAAGGC CCAGTCTTCC GACTGAGCCT TTCGTTTTAT TTGATGCCTG GCAGTTC-
CCT ACTCTCGCGT TAACGCTAGC ATGGATGTTT TCCCAGTCAC GACGTTGTAA AAC-
GACGGCC AGTCTTAAGC TCGGGCCCCA AATAATGATT TTATTTTGAC TGATAGT-
GAC CTGTTCGTTG CAACAAATTG ATGAGCAATG CTTTTTTATA ATGCCAACTT TG-
TACAAAAA AGCAGGCTCC GCGGCCGCCC CCTTCACCCC AGCCAAGACA TTGT-
GATCTT CAACCTCCAT CCAACCCTCT GAGTCTTGTC TGCTATAAAC TTGGATCAAA
GCCTCTCCAT CTGAATGTGT CTCATTCAAG GCGTGTGAAA CATTCGTCAC GAG-
CATATAC GTATCCTTGT CGTTGGTTAA CCCTCTAAGA GTAGAAGTGA TCACTGTAAG
AGGATATGTC CGCTGGTGCA CGACTCGATT CACCGGTTTA CCCGATTCAG AAGT-
CACCTC CACGATCCGT TCGGTCTTCA CTCTCTGCTC AACCCTCTTG TATGTTCCGT
TTTTCTCGAA CCTCACCAAG CTGTCAACCT TGAACATGCT CTTGACCATG GTG-
GTCAGAT TACCTTTACT TGATCTAACC CAGCCATCAT ATTCACTACT TACCTCTGCT
TCAACCTTAA ACGACCCATC AAGCTGTTCA AGCTGTTCTT GTCTCATCAT ATGCC-
GACTC GGGCTATCAT AAAGTCTAGA CCCAGCTTCA ACATTCGAAG ACCCGTGATC
TAACCAGAGG TGCAAATTAG CGTCCACAAG CCAGTATGAT ATCCCATCGT TGACCC-

CAAA CGCAAACTCA TGAGACTTTC CATCTAAAAG CAACCCGAGA AATGGCGTCA
AGTCCATGTC ATAGGAAGGT AGATTGAAGG CACCGATTGC TACAACTGGT TCCCA-
GAACA AGGGATTGAT TCCACCAGTA AAGATCACCG GAAATGGCAC CTCGGATCCC
ACGTATCTCC CATCTATCTT AACAAAAACT TCCCGGTATG CACCATTGCC ACGCC-
CGGTT GTTAGATTGT TCGTTCTGAT ATAAGAATTT GGCGGGTTTG AATACCAGAA
CTCATCGTTT CCATGGAACG ATACATATAG TTCCAGCACA ATCTGACGAG TGTTG-
GACGG AATTTGAATT CCTTTTGAGT AAGTTTCTCT GGGGTTCTCG ATCATGAACC
AGAACCCTCT GTTCCCTCCG TCACATACCG GAATTATCAA ATCCGCAGGG GTTTGAT
CTC TTTTCTGGGA ATCCACAAAC CCTAATCTGT TACTAATCTT CAAATTTGAC GCAAT
AGGAT TGAATTCGTA GAAGATGAGG GTGACATTGA TATGATAGAT GCCTGTGTAA
ACATCGTTGA CTATGTTCTC AAGCATCATT GTGACATTTA AATCGGATCT CATAAA-
GAGC GAGGAATACC TAGAGACGTC TTTCCGAACA TTCCAGAAGA TTCCGGATGG
ACTCGGCTCC GCCGTACTGG TGCGAAGTAG CTCCACTCCG CCGAGCCACA AGC-
CGGAGAT ACGATCGTAC TGATCGCCAC TCGAGGCGGC GCGTAGGTCA AGTACAA-
CAT AAGACCACGG CGGCGATATG CAGCTGGAAG GGGGAGTGTA TGGAGTGGTA
AAAGGAGGTC TGTTGATGGT GTTGGCGAAT GAATGGCGGA AGAGGACATG CGAG-
CATGAC GGCGTTAGCT GATCGGACGG TAGGGGCCGC CTGAGCTCCT CGTACTC-
CTG AGGCTCGACC GGTGACCGGA GACAAGTCGA AGCCGATCTA AGGAATCGAT
CCGGCGAGGA AGGACGAGAA ACGGCGGTAG CAGTGAGAAA AGCAAGTGTG AA-
GAAGAACT TAACGGCGCC GTTTTGATTT TCTGGCATga tagcttctgtgtttttgatt atccgagctc
gccgatgcga acttcaatgg agactggaga gagagaagggacgtaattaa acaccaaagc gtttttatat ttcggagaat caactacgtg
tcgtttgaca tcctaaattg gcagatagta gaaaggaaAT GTTGAGACTG GAGAGTTTGA TAG-
TAACAGT GTGGGGACCA GCGACGgtaa cggaattcga aatttatttt agcgtcacgt gtggttacttataatctgtg
cacgtgtcaa aataagagag ggctactagt gacactttcg tcactgtcacttaattttag cctttctcct gacatgctta ctctctcctc
tcccgctacg atcactgcgagttttccctt cttaagctct catggcggct tcataaatct atatacagac aaatttacacataaagaaac
gcattaaaaa aactatttac tcagattcag cttcttctcc acgcaaagac tcgcttctct ttttttctct tcctgagttt ggattttttt
tgcttgagct gaatcatgttttattctcac cagCTTCTAG CTCGAAAAGC TCCGTTGGGT CAGATATGgt
gagtcaaattctatcagtga aacgttttcc gattatcacg atgattttgc aacctctgtt tctgtatctgtttttgaaat cggctgggaa
cttccttgtc gcgatcaagc acttgtcact gcttcaaatcgtcactgcga tttccattga ttgatctgta tatggattat tagtgaatgc
atagagtaaa atctctaaac tctgtttttg atttttcttc ttcttaagGA TGGCCGCTAC ATTGCACGCG AAGAT-
CAACC GGAAGAAACT AGATAAGCTC GATATCATTC AAATCTGgtg agaggagatg agatagtgtt
cgcccttttt ttgggtttta ctctgcttcg atttttttgg tgttcctgccatgaaaacga acgttgattt ttccattttt gttggttact
ttactcttac agCGAAGAGATTTTGAATCC GTCGGTTCCG ATGGCTCTTA GACTCTCCGG
GATTCTTATG Ggtatgcttttgctcatcgg attttttttt tcctcagttc ttcacattct gggttcttca atgttttgtttattcgatag

GTGGTGTTGT GATTGTTTAT GAGAGGAAAG TGAAGCTCCT ATTCGgtaattttctgattc aaatcatttt
tgaattttgg gatttaagtt tacactctcc tctttttctg atgagactct gctctttttc tgggttttat tttccgtcaa tatttcagAT
GATGTTAATC GCTTTCTGgt aatcaaatct tctccgatcc tcttctattt tttgtttccc cgtcaaaatc atattcacgt
acctcgattt ctggattttc acatgtgttt atttttctca gGTTGAAATT AATGGAGCTT GGCGCACAAA
ATCTGTTCCG GATCCCACTT TACTACCTAA AGGAAAAACC CATGCCAGgt aatgtcacat
cttcttcctt gaaggagctt ctcatgccat tgaaggattctgaaggatca aatcgtaaac ataagaacat tgtcgtgaag aataatagaa
atgattggcgtgttggattt ttttgtttgc agGAAAGAGG CTGTTACATT GCCTGAGAAC GAA-
GAAGCTG ATTTTGGAGA TTTTGAACAG ACTCGTAATG TTCCTAAATT TGGCAATTAC
ATGGATTTTCAGCAGACTTT TATTTCCATG gtaatcaact caattccctg tgaattctag attgaattgggtatttattt
tctttctacg tctcaatatt catcaattcg tagaattgtg gcttgaactc tccaacctaa aacagtgtat aagatataga aaaatcagta
gccatcattc ttcattgcccataaaagtgg agtaaataag ttggcgcata aagggcacct aagacttggt ctaactcaag catgtggttt
atgagtagcc tcattttagt ctacaggtga atatttcaaa ggaaaatctt tttggaactt gaagcatatg aactcaaacc aaaatcattt
gaacatcgct taagattttgacgtgtaacc tatgggctgt ggaaatggac aatgcacaaa agatgcaact gttaatgtga attgatcata
gtagtgttca tctccttgta taccaatgat tactcgtgtg catcttcagt tgatctaatt cattgcgtgg ttttatctct ttgcagCGGT
TAGATGAATC CCATGTTAAC AATAACCCCG AGCCAGAAGA TCTTGGACAG CAGTTC-
CATC AAGgtgagta tcttgagaca aaacctttgt gagtatacga catgttaata acttacattt atccgcttgt caatttctgt
aacattctca gCTGATGCCG AGAATATCAC ACTCTTTGAG TATCATGGTT CATTCCAGAC
CAACAATGAA ACATATGATC GTTTTGAAAG gtgagctttc aatcccacta tctatcttcagtctttaatt
cccagatttt aaaccttctc gaataactgt tgaaggtaga agttgtagcg tttcactatt tctgtatttt ctttcatcca cctttcagca
aaaagctggt gatgatttgt tctattctat tctgcttttt aatggcatct cagATTTGAC ATCGAAGGAG ATGAT-
GAAACACAGATGAAC TCCAATCCAA GAGAAGGCGC TGAAATACCT ACAACTCTCA
TCCCATCACCACCTCGTCAT CATGACATTC CCGAAGgtag atagttatgt cactcttccc ttctataatc
gtgacatctg gttattgtat ggggatatga atttttgagt caaagatgaa attgttttca atttttaaat agtgttaagt ttatgaagcc
aataacaatg acttgggcga gtctcttgta taataatcat gtatgtgacc tgggaatgta tttttgttga agcttctgaa tgatatgaac
actgagcttc gtgaacagag cagaacaaat ttactaatct ttagccatct gcagGAGTCA ACCCCACAAG
CCCTCAGCGC CAGGAGCAAC AGGAGAATCG TAGGGACGGA TTTGCTGAGCAGATG-
GAGGA ACAAAACATA CCGGACAAAG AGgtattttc acttactagg aataccctca tctagtagaa aatacattga
attgggtttt gttctgcatg cagGAACACG ATAGACCACAACCAGCGAAA AAGAGAGCAA
GAAAGACAGC TACTTCAGCG ATGGATTATG AGCAAACTAT TATCGCTGGT CATGTT-
TACC AGTCATGGCT CCAGGATACT TCTGACATTC TCTGTAGGGGGGAAAAGAGA
AAGgttcaaa tttttgaact cattggcctt tattgtatta actggtgctc atgttggcct tacatctgac atctttttgt agGTTC-
GAGG AACTATCCGG CCAGACATGGAAAGTTTCAA ACGTGCGAAT ATGCCACCTA
CACAACTCTT TGAAAAGGAC AGTTCTTACCCGCCTCAGCT TTACCAGCTT TGGT-
CAAAGA ATACTCAAGT TCTTCAAACC TCATCATCTG gtctctctct atccataact gttggttttt

tgttttctcc ttgttgaaac gaatctgaca tttggaaatg tcgatacagA ATCTCGACAT CCTGATCTCC GT-
GCGGAACA ATCTCCAGGG TTTGTTCAGG AGAGAATGCA TAACCACCAT CAAACA-
GACC ATgtaagtcg cggaagcata ttctttcctg aaagtttagt atcaataaga gaaaggcaca tttcatattc tctctaacagaatgtggatt
tatgcagCAT GAGCGCAGTG ACACAAGCTC CCAAAATCTT GATAGTCCCG
CAGAAATACT CCGGACAGTT CGTACTGGGA AAGGTGCTTC AGTAGAAAGC ATGATG-
GCTGGATCTCGAGC AAGCCCTGAA ACTATTAACC GCCAGGCTGC TGATATTAAT
GTCACGCCATTCTATTCTGg ttaaaagctt ctctatttta tccagtcttc agatcatagc ttggtattgt ggaatattta
gcgtaagtta tttgattttc atgggaatta cagGAGATGA TGTGAGATCC ATGCCTAGTA CACCATC-
CGC ACGTGGAGCA GCTTCAATTA ACAACATAGA GATCAGCTCT AAAAGgttaa gaacacatct
aagtttctct gatactaaaa ccaatagtaa cttacaaacagttttccat tggaagacga tcactaaatc ttcttctgct taccaaatca
gTCGCATGCCCAATAGAAAA AGACCAAATT CCTCACCAAG AAGAGGACTC
GAACCAGTGG CGGAAGAGAGACCGTGGGAG CACCGTGAAT ATGAGTTTGA GTTTTC
AATG TTACCTGAAA AACGCTTCACAGCCGATAAA Ggtagaagtt tttaattgtc ttttgatcct atacaacttt
caaaaacacc atcttacaaa ccttaacgtg gcttcttttt cacagAAATA CTATTTGAAA CTGCATCTA-
CACAGACTCAA AAGCCAGTGT GCAATCAATC AGACGAGATG ATAACAGATA GCAT-
CAAAAGgtaagagatt caaaattatc tgccaaatct atatactgag taacaatgat gagatctataaacgaaacac agTCAC-
CTGA AGACACACTT TGAAACACCT GGAGCTCCTC AAGTGGAATCTCTTAACAAG
CTCGCTGTTG GAATGGACAG AAACGCTGCA GCTAAACTCT TCTTCCAATCCTGTGgtact
aacttctact tcaacttaat ttcactttct ttgagcataa aaaaacctgactatgattat tgtttatatg tccagTGTTA GC-
TACTCGCG GAGTCATCAA GGTAAACCAAGCAGAGCCTT ATGGGGACAT TCTCATTGC
A AGAGGACCCA ACATGCCCGG Gtgtacaggagtaccgcccc gtccggtcct gcccgtcacc gagatcagcg
gagagttcat ggtgtccaagggcgaggagc tgatcaagga gaacatgagg atgaaagtcg ttatggaggg aagcgtgaacggtcaccagt
tcaagtgcac cggcgaggga gaggggcgtc catacgaggg agtccaaactatgaggatca aagtgattga aggaggccca
ctccctttcg ccttcgacat cttggctacctctttcatgt acggctcccg caccttcatc aagtaccccg ccgatatccc
ggacttcttt aagcagtcat tcccagaggg tttcacctgg gagagagtca cacgttacga ggacggcggagttgtgaccg tcactcag
ga caccagcctg gaggatggcg agctggttta caatgtgaaagtccgcgggg tgaacttccc ctccaacgga cctgtcatgc
aaaagaagac caagggctgg gaaccgaaca cggagatgat gtatccagcc gacggtggcc tcaggggata caccgacatt
gcactcaaag tggacggcgg cgggcacttg cattgcaact tcgttaccac ttaccgctcg aagaagaccg tcggaaatat caagatgccc
ggagtgcacg ccgtcgatca ccgtcttgag aggatcgagg agagcgacaa cgaaacctac gtggttcagc gcgaagtcgc
tgtggccaagtactccaacc tgggtggagg catggacgag ctttacaagt gaCCCGGGTA Aggtttgatttctaaattat
aaaagattct ggtgaaccga ttatccatag ttgttttgct tttcatattctagcagagag agttcgtaga cttttttaag ttataaagag
caagcgttct ttacccaaattcctctgttt ggtccttttg ttatatggtt attagtacct catacatcat cacatcctagctttgtccga
aaacatctga aactaatttt tacattattc tataatttaa gtattttactcgcatatatt gagtcttctt agaagatgta ttgaacagag
cataaacaaa acaatagtttaatatatact ccgttgacaa agaaaatggt ggcagtcacg taataagccg caactgcccc-

catttcatac aatacatgta taatcttcat aattagtcgc tcacacgttt catacaatttgtcaaatttc tcaaaaaatt aaactaatta
cacaatcaaa taccagcctc tatatgtttctatatatacg catatttctt acccctgaat cacacaAAGG GTGGGCGCGC
CGACCCAGCTTTCTTGTACA AAGTTGGCAT TATAAGAAAG CATTGCTTAT CAATTTGT
TG CAACGAACAG GTCACTATCA GTCAAAATAA AATCATTATT TGCCATCCAG CT-
GATATCCC CTATAGTGAGTCGTATTACA TGGTCATAGC TGTTTCCTGG CAGCTCTGGC
CCGTGTCTCA AAATCTCTGATGTTACATTG CACAAGATAA AAATATATCA TCATGAA-
CAA TAAAACTGTC TGCTTACATAAACAGTAATA CAAGGGGTGT TATGAGCCAT ATTC
AACGGG AAACGTCGAG GCCGCGATTA AATTCCAACA TGGATGCTGA TTTATATGGG
TATAAATGG G CTCGCGATAA TGTCGGGCAA TCAGGTGCGA CAATCTATCG CTTG-
TATGGG AAGCCCGATG CGCCAGAGTT GTTTCTGAAA CATGGCAAAG GTAGCGTTGC
CAATGATGTT ACAGATGAGA TGGTCAGACT AAACTGGCTG ACGGAATTTA TGC-
CTCTTCC GACCATCAAG CATTTTATCC GTACTCCTGA TGATGCATGG TTACTCACCA
CTGCGATCCC CGGAAAAACA GCATTCCAGG TATTAGAAGA ATATCCTGAT TCAGGT-
GAAA ATATTGTTGA TGCGCTGGCA GTGTTCCTGC GCCGGTTGCA TTCGATTCCT
GTTTGTAATT GTCCTTTTAA CAGCGATCGC GTATTTCGTC TCGCTCAGGC GCAAT-
CACGA ATGAATAACG GTTTGGTTGA TGCGAGTGAT TTTGATGACG AGCGTAATGG
CTGGCCTGTT GAACAAGTCT GGAAAGAAAT GCATAAACTT TTGCCATTCT CACCG-
GATTC AGTCGTCACT CATGGTGATT TCTCACTTGA TAACCTTATT TTTGACGAGG
GGAAATTAAT AGGTTGTATT GATGTTGGAC GAGTCGGAAT CGCAGACCGA TACCAGG
ATC TTGCCATCCT ATGGAACTGC CTCGGTGAGT TTTCTCCTTC ATTACAGAAA CG-
GCTTTTTC AAAAATATGG TATTGATAAT CCTGATATGA ATAAATTGCA GTTTCATTTG
ATGCTCGATG AGTTTTTCTA ATCAGAATTG GTTAATTGGT TGTAACACTG GCAGAG-
CATT ACGCTGACTT GACGGGACGG CGCAAGCTCA TGACCAAAAT CCCTTAACGT
GAGTTACGCG TCGTTCCACT GAGCGTCAGA CCCCGTAGAA AAGATCAAAG GATCTT
CTTG AGATCCTTTT TTTCTGCGCG TAATCTGCTG CTTGCAAACA AAAAAACCAC
CGCTACCAGC GGTGGTTTGT TTGCCGGATC AAGAGCTACC AACTCTTTTT CCGAAG-
GTAA CTGGCTTCAG CAGAGCGCAG ATACCAAATA CTGTCCTTCT AGTGTAGCCG
TAGTTAGGCC ACCACTTCAA GAACTCTGTA GCACCGCCTA CATACCTCGC TCTGC-
TAATC CTGTTACCAG TGGCTGCTGC CAGTGGCGAT AAGTCGTGTC TTACCGGGTT
GGACTCAAGA CGATAGTTAC CGGATAAGGC GCAGCGGTCG GGCTGAACGG GGGGTT
CGTG CACACAGCCC AGCTTGGAGC GAACGACCTA CACCGAACTG AGATACCTAC
AGCGTGAGCA TTGAGAAAGC GCCACGCTTC CCGAAGGGAG AAAGGCGGAC AG-
GTATCCGG TAAGCGGCAG GGTCGGAACA GGAGAGCGCA CGAGGGAGCT TCCAGGG
GGA AACGCCTGGT ATCTTTATAG TCCTGTCGGG TTTCGCCACC TCTGACTTGA GCGT

CGATTT TTGTGATGCT CGTCAGGGGG GCGGAGCCTA TGGAAAAACG CCAGCAACGC
GGCCTTTTTA CGGTTCCTGG CCTTTTGCTG GCCTTTTGCT CACATGTT
ASY3 −TagRFP
pENTR-progASY3-gASY3-TagRFP 3’UTR
ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctgataccgctcgc cgcagccgaa cgaccgagcg
cagcgagtca gtgagcgagg aagcggaagagcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgctagccaggaa
gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgcttagtttgatgcc tggcagttta tggcgggcgt cctgcccgcc
accctccggg ccgttgcttcacaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctggcagttccct actctcgcgt taacgctagc
atggatgttt tcccagtcac gacgttgtaaaacgacggcc agtcttaagc tcgggcccga gttaacgcta ccatggagct
ccaaataatgattttatttt gactgatagt gacctgttcg ttgcaacaaa ttgataagca atgcttttttataatgccaa ctttGTATAG
AAAAGTTGTA cgaattattt cttcgttgat atcgtatataaaatttaaaa ctcatagaat ttctattcaa aaagtaattg gtgattaaat
gaatggagtg cctatatatg tatttttttt ttcttgcaat caattttgat ttagaatttg catgcatgat atttttttaa tattttttta
tcctttaata attttttttt atattatcta aagttgtatt tgtattcctt acataaagtt tggttacttt catctctaag gtaaactgct aatattaaag
aaagtaattt aatggaagca aaaagtgtta aaatatgtaa tataatttga atggaagagt agcccccgag ggcaatcaag
tggaggtctt ccatggttag gatgcagaag cttaatacgt ttatatagat agataaacaa tagcctccat aaagtaaaag atacttgcat
gcgtgcgtgt aggtattgag tcgagatgtt acgtgcgtgt ctttttatag atactatata ttattggttc ttctaaatcc ttaaaaatgt taagattacg
cacgcaattc atgaattgta tatagattta ccaaatcttg aattgatttt aaatttctta caaagcaaat acactggtag tgtgacattt
tgtccctgtg gattctgtat gacgtaatct cgttcctcac attatgacac ttaatcgagt gttttttttt ccctatcatt ttttttgtaa atttatcctc
tacaaattat gtatgtttgt gaactattaa atacgatgtt gatatatata tatatatata tataaactat ataataaata agtgaattat

tttacgagta tggatcataa ataaaacatg gaataacaat agaatgtttg ttaaatagaa ctcaaattaa tgatcagtta tgtacagtcc
ggccttaggc ccaggcacta cccacttggg ccgggcctgg ttatgtagca tatatataaa cgctagtcac cgggatagtt ggacttggat
ggctctagcc tgacccgtca aagtttaacg gaaacttgac atatgcaaag accgtctccg tttgaacttt gaagcgattg
aaattttttg agaggcgaat taaggcgcca aaattttgag aatgataagt agattacgac acatgtactc acctcaatct gacggttacg
atgcaatgta aatacgaaga aatctcaggg ttaaaatcgt ccaaatgttc ggcgtcgtgt atacgcaatt cagctacaga cagtgtttgt
tacacaccat tttgagaaat ctaaattttc attttccccc gcttctctcc tagaaatttc gaatttctgc ttctctctct ctctatcagt tttctcagat
cttattccag ttaatattcc ggcgatcttc tttctctctt tgatcactta attcttcacc ggaatcagag agtcagggct ttcgcacttg
tacccgcgag ctttgattag gtaatttctt agccaatgat accatcgctc gattttctct gttttcgttt tcgattcatg gctcgttact
ctcatattca gtgataagtc gtcgcgatct atgccccctt tcgttaaatt ggtgttatcg tctcgatttt gagtttagta ccagcgttat
agtgttgtct ttgcataatc ggagagtttt ggtatgaaat tcgttgctag agatatcaca tctcgctagt tgttcatcgt ttcaagcatc
tattatctgc tgcttctctt gttttcgtca tattatccag atctagcacc ttcttatttt gggattttct attaccaaat tttttatccg agtgttagaa
atttaccatt cctaatgatt aatttatcat ccattatcgc tgaatatagc ctgcgttgta ctgctttact tctctgtttg tgttgctatt
cagaaattat atgagtgatt aggtgttcgt tgatatttac attctttttc ccagcagaat tgttaacctt gtggtgatac atttcaggaa
aagATGAGCG ACTATAGAAG CTTCGGCAGT AACTACCATC CATCAAGTCA ATCTA-
GAAAG ATTTCTATAG GAGTTATGGC TGATTCACAA CCGAAAAGGA ATCTTGTGCC
AGATAAGGAT GATGGAGATG TTATTGCTAG AGTAGAAAAG TTGAAATCAG CCAC-
CGTAAC TGAATTACAG GCGAACAAGA AAGAGAAAAG TGATTTAGCT GCCAAG-
CAAA GGAATTCAGC TCAGGTCACA GGACATGTGA CCTCACCTTG GAGGTCTCCG
AGATCGTCTC ATCGGAAATT AGGGACTCTT GAGAGTGTTC TTTGTAAGCA AACATC-
TAGT TTGTCTGGTA GCAAAGGATT AAACAAGGGA CTTAATGGAG CACATCAGAC
ACCAGCTCGT GAATCATTTC AAAACTGTCC CATTTCGAGT CCTCAGCACA GTCTTG-
GTGA GCTGAATGGT GGCAGGAACG ATAGAGTGAT GGATAGGAGT CCAGAGAGGA
TGGAAGAACC TCCATCTGCA GTCTTGCAGC AAAAGGTTGC CTCACAGAGA GAGAA-
GATGG ATAAGCCAGG GAAGGAGACA AATGGAACTA CTGATGTTTT GAGATCAAAA
CTGTGGGAGA TATTGGGAAA AGCTTCACCA GCAAATAATG AAGATGTGAA CTCT-
GAGACC CCAGAAGTTG AGAAGACCAA CTTCAAGTTG AGTCAAGACA AGGGTTCAA
A TGATGATCCT CTTATTAAGC CTAGACACAA TTCAGATTCT ATCGAAACTG ATTCT-
GAAAG CCCTGAAAAT GCTACCAGAA GGCCAGTAAC CAGATCTTTG TTGCAGAGGA
GGGTAGGAGC CAAAGGTGTC CAGAAGAAAA CCAAAGCTGG TGCCAACTTA GGTCG-
CAAGT GCACAGAACA AGTAAATAGT GTATTTTCTT TTGAGGAAGG TTTGCGCGGA
AAAATTGGCA CTGCTGTGAA TTCCAGTGTT ATGCCAAAGA AACAAAGGGG TAGAA-
GAAAA AACACTGTTG TAAAATGCCG TAAGGCTCAT TCTCGAAAAA AGGATGAAGC
TGATTGGAGT CGGAAGGAGG CGAGCAAGAG CAATACTCCA CCACGTTCTG AAAG-
CACAGA AACTGGCAAA AGATCTTCAT CTTCAGACAA AAAGGGAAGT TCCCAT-

GACC TTCATCCACA GAGCAAAGCC CGGAAACAGA AACCAGATAT TAGCACAAGG
GAAGGAGATT TTCATCCATC GCCAGAAGCT GAGGCAGCAG CTCTGCCAGA GAT-
GTCCCAG GGATTATCTA AAAATGGCGA TAAACATGAA CGGCCGAGTA ATATTTTCAG
GGAAAAGTCT GTTGAGCCAG AAAACGAATT CCAGAGTCCA ACCTTTGGAT ATAAAG-
CACC AATCTCAAGT CCTTCCCCAT GTTGTTCTCC AGAAGCATCT CCTTTGCAAC
CTAGGAATAT TAGTCCCACA TTAGATGAGA CGGAAACACC AATATTTAGC TTCG-
GTACTA AGAAAACCTC TCAAGGGACA ACAGGCCAAG CGTCAGATAC AGAAAA-
GAGA TTGCCTgtaa ccttcataaa ctgaagcggc tttatctata cttaagttgc atattgactt tatatactcc tctttgactt
ttgtgtttac ttatttattt tcagGATTTC TTGGAGAAGA AAAGAGATTA TTCTTTCAGA AGA-
GAAAGTT CTCCTGAGCC AAATGAAGAT TTAGTTCTGT CTGATCCGTC GTCTGAT-
GAG CGAGACTCAG ATGGTTCAAG AGAAGATTCA CCAGTGTTAG GCCATAACAT
CAgtatgctt cttaattctt atcctagacc atataaaaac tggaggatct tattttcttc ttaacagaat ccatgttttg cttattctat
tgatcttctg attcagGCCC CGAAGAAAGA GAAACTGCCA ATTGGACTAA TGAGAGATCT
ATGTTAGGCC CTAGCTCAGT TAAACGGAAC TCTAACCTTA AGGGCATTGG ACGT-
GTCGTG TTAAGTCCTC CCTCCCCTTT GTCAAAAGgt ataatgctca tttgtatgtc actgttgtgg
gtttatatac tgcaccctca tttgttttgt cctggaaact gagcagGGAT CGATAAAACT GATTCCTTCC AG-
CATTGTTC AGAGATGGAT GAGGATGAAG ATGAAGGCTT GGGAAGgtct cttgtagata taactaatat
tacgtgtcta tctttaatct ttgttcctgt cttaagttga ttatttacca tgtctcagGG CTGTTGCATT GTTCGC-
TATG GCTCTTCAAA ACTTTGAGAG AAAACTGAAA TCTGCAGCTG AAAAGAAGTC
CTCAGAAATT ATAGCATCAG TCTCCGAGGA GATACATCTG GAGTTGGAGA ATAT-
CAAGTC CCATATCATA ACAGAAGCgt atgtttgtat aatttattct gcattctttc ttttaaatta tcaatcaaag
atcttgttaa ttatggtaca gGGGAAAGAC AAGTAACCTA GCCAAAACTA AGAGAAAGCA TGC-
CGAAACA AGATTACAAG gtacatcaac acattatatc ttaatagtta atactgtact cgaattcaca gtcatgaaca ttaagaaagc
ttagaagttc aaatatacta tctttgcagt ttaatcctga ttcaaggtag tagatcggaa atttgatatc aagtattact ctttagattg
catttagtct tgaagtactc accatcttgc ttcattcttg tgctttcatg tctctttgat tagacacaca cacacaccta actatacttt
cggctctaac atagacatga aacttgcatt taacatagtt atatcaagac aaagctaaat gctcaattca ttcctatgtg gagaataata
aaaaattgag ctgtgtattg ttgtggaaca tgagagtctg gaaatgtctc catatttcca ctcattgaat aaaaatatgc aagatccgtt
ctgatctatt gtgttcactg ataatatcta atcgtctaac agAGCAAGAA GAGAAAATGA GAATGATCCA
TGAAAAGTTC AAGGACGATG TCAGCCATCA TCTTGAAGAT TTTAAGAGTA CTATC-
GAAGA GCTTGAAGCA AACCAGTCAG AGCTGAAAGG AAGCATAAAG AAGCAAAgta
tgttaacttc tgtaattcct gcttttacta caacgaattc taacactaga aatacttgaa acatatgatg ttatcaaagt tagattcttc atggcaagat
agttttaata taataatagc aggaggacaa gtatttccta aactcacttg ataacttttt atctctctat gacagGAACA
TCACACCAAA AACTCATTGC ACATTTTGAA GGAGGCATCG AGACAAAACT GGAC-
GATGCG ACCAAAAGAA TCGATTCTGT AAACAAGgca agtcattgtt ctgcttctta agagtcagat tc-

taaaaatg gttaaatcat gaattcaaaa gcacaactta aaaatgacgt attggtttac agTCTGCGAG AGGAAAGATG
CTGCAGCTGA AGATGATTGT CGCAGAATGT TTGAGGGATG ATCCCGGGgg tggcggtgga
tcaggcggag gtgggagtgg tggtggcgga tctATGGTGT CTAAGGGCGA AGAGCTGATT AAGGA-
GAACA TGCACATGAA GCTGTACATG GAGGGCACCG TGAACAACCA CCACTTCAAG
TGCACATCCG AGGGCGAAGG CAAGCCCTAC GAGGGCACCC AGACCATGAG AAT-
CAAGGTG GTCGAGGGCG GCCCTCTCCC CTTCGCCTTC GACATCCTGG CTACCAGCTT
CATGTACGGC AGCAGAACCT TCATCAACCA CACCCAGGGC ATCCCCGACT TCTT-
TAAGCA GTCCTTCCCT GAGGGCTTCA CATGGGAGAG AGTCACCACA TACGAA-
GACG GGGGCGTGCT GACCGCTACC CAGGACACCA GCCTCCAGGA CGGCTGCCTC
ATCTACAACG TCAAGATCAG AGGGGTGAAC TTCCCATCCA ACGGCCCTGT GATGCA-
GAAG AAAACACTCG GCTGGGAGGC CAACACCGAG ATGCTGTACC CCGCTGACGG
CGGCCTGGAA GGCAGAACCG ACATGGCCCT GAAGCTCGTG GGCGGGGGCC AC-
CTGATCTG CAACTTCAAG ACCACATACA GATCCAAGAA ACCCGCTAAG AACCT-
CAAGA TGCCCGGCGT CTACTATGTG GACCACAGAC TGGAAAGAAT CAAGGAGGCC
GACAAAGAGA CCTACGTCGA GCAGCACGAG GTGGCTGTGG CCAGATACTG CGAC-
CTCCCT AGCAAACTGG GGCACAAACT TAATggaggt ggaCCCGGGT GAACAAGTTT
gtacaaaaaa gttgaacgag aaacgtaaaa tgatataaat atcaatatat taaattagat tttgcataaa aaacagacta cataatactg
taaaacacaa catatgcagt cactatgaac caactactta gatggtatta gtgacctgta gaattcgagc tctagagctg
cagggcggcc gcgatatccc ctatagtgag tcgtattaca tggtcatagc tgtttcctgg cagctctggc ccgtgtctca aaatctctga
tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa
tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgtt
gc caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga
tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa
atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc
tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct
ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg
ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc
ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg
atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg gcagagcatt acgctgactt gacgggacgg cgcaagctca
tgaccaaaat cccttaacgt gagttacgcg tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt

ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
gaacgaccta caccgaactg agatacctac agcgtgagca ttgagaaagc gccacgcttc ccgaagggag aaaggcggac
aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgtt
TagRFP− CENH3
pENTR-proCENH3-TagRFP-CENH3 3’UTR
CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT ACCGCCTTTG AGTG-
AGCTGA TACCGCTCGC CGCAGCCGAA CGACCGAGCG CAGCGAGTCA GTGAGC-
GAGG AAGCGGAAGA GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG
ATTCATTAAT GCAGCTGGCA CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCG-
CAAC GCAATTAATA CGCGTACCGC TAGCCAGGAA GAGTTTGTAG AAACGCAAAA
AGGCCATCCG TCAGGATGGC CTTCTGCTTA GTTTGATGCC TGGCAGTTTA TGGCGGG
CGT CCTGCCCGCC ACCCTCCGGG CCGTTGCTTC ACAACGTTCA AATCCGCTCC
CGGCGGATTT GTCCTACTCA GGAGAGCGTT CACCGACAAA CAACAGATAA AAC-
GAAAGGC CCAGTCTTCC GACTGAGCCT TTCGTTTTAT TTGATGCCTG GCAGTTC-
CCT ACTCTCGCGT TAACGCTAGC ATGGATGTTT TCCCAGTCAC GACGTTGTAA AAC-
GACGGCC AGTCTTAAGC TCGGGCCCCA AATAATGATT TTATTTTGAC TGATAGT-
GAC CTGTTCGTTG CAACAAATTG ATGAGCAATG CTTTTTTATA ATGCCAACTT TG-
TACAAAAA AGCAGGCTCC GCGGCCGCCC CCTTCACCgc ttctccaatc taccactaat ttcacccttttcgtaccatt
ttctccatga tccgtcaatt ttgatcccaa ccatcgattt agggttttttcccccaaatc tctgtttaac gattgat-

tat aattgatttt ttttagGTTT GGTCACTTGT ACGGAGATTT GATCAGCCGC AGAAATACAA
ACCGTTTGTG AGCAGATGTA CAGTAATCGG TGATCCTGAA ATCGGCAGTC TTAGA-
GAAGT CAATGTTAAA TCTGGTCTTC CTGCAACAAC ATCTACTGAG AGATTAGAAC
TTCTTGATGA TGAAGAACAC ATCCTCGGTA TCAAAATCAT CGGTGGTGAT CACA-
GACTTA AGgtacagcg agagaaatca aatctcattg atgtttgttt atatgcagat tctcattgat tgtttgtttg cagAAT-
TACT CGTCGATTTT GACGGTTCAT CCGGAGATAA TCGAGGGAAG AGCAGGAACG
ATGGTGATTG AATCGTTTGT AGTTGATGTT CCTCAAGGTA ACACAAAGGA TGA-
GACTTGC TACTTTGTTG AAGCACTTAT CAGATGTAAT CTCAAGTCAC TAGCAGATGT
TTCTGAAAGA TTGGCTTCTC AGGACATTAC TCAGTGAact acataatcaa tgaacaaggg cattgaag
tg aagtatcaat tccagtttgt gatataatca atattcttca ggattttttt ggtttggcct agatatatat atagatatct atcctcggta
atgaccagtc taaaaagatg tacatattgt cccaatggtg aagttttgat gtaagatatc tcctggtggt ttgttatttg tagatatttt tgtaaacaat
gtaaatgtga atggtttatg atgtataata tatagttcac aaaagatgtt tctgtagact tcagattcca cttctctatt gaacagaacc
tatgattgga tgctgagaac ttgtaaagaa tctgaggcag aaagttgaaa aactgtgtca atttcattaa cttgaaaaga
tgagcataaa ttgggagaga gagagagaga caaagatttt gaattgaggt ttaacggtaa aacacacaaa acctattccc ctctgtttcc
aattttcatc taaacaaaca ggtacatatt tgaatgtaat attgtataca gaccaggggt aaaacaggaa ctaaagaagg
ctaacaatcg agtcgaaccc tctatgtgaa gccacaggtt tagtgcaaat tgtaataagt tgttcagaga gactcttgac tgaaacaaat
tgtgaagcag attcgatttt aaaatcaaaa tttgagtgtc gagcgggaaa gtaaaagttc cgctccaatc ttctaatctt
ttcgtatcta gcgggaaatt tctcagcagg tgactttcat aatcgcagtt ttcgtcgatt ctcttttccg attttacgat tcctctctct ctctcatggt
gcgatttctc cagcagtaaa aatcaCCCGG GGGTGGCATG GTGTCTAAGG GCGAAGAGCT
GATTAAGGAG AACATGCACA TGAAGCTGTA CATGGAGGGC ACCGTGAACA ACCAC-
CACTT CAAGTGCACA TCCGAGGGCG AAGGCAAGCC CTACGAGGGC ACCCAGACCA
TGAGAATCAA GGTGGTCGAG GGCGGCCCTC TCCCCTTCGC CTTCGACATC CTG-
GCTACCA GCTTCATGTA CGGCAGCAGA ACCTTCATCA ACCACACCCA GGGCATC-
CCC GACTTCTTTA AGCAGTCCTT CCCTGAGGGC TTCACATGGG AGAGAGTCAC CA-
CATACGAA GACGGGGGCG TGCTGACCGC TACCCAGGAC ACCAGCCTCC AGGACG-
GCTG CCTCATCTAC AACGTCAAGA TCAGAGGGGT GAACTTCCCA TCCAACGGCC
CTGTGATGCA GAAGAAAACA CTCGGCTGGG AGGCCAACAC CGAGATGCTG TACCC-
CGCTG ACGGCGGCCT GGAAGGCAGA AcCGACATGG CCCTGAAGCT CGTGGGCGGG
GGCCACCTGA TCTGCAACTT CAAGACCACA TACAGATCCA AGAAACCCGC TAA-
GAACCTC AAGATGCCCG GCGTCTACTA TGTGGACCAC AGACTGGAAA GAATCAAGG
A GGCCGACAAA GAGACCTACG TCGAGCAGCA CGAGGTGGCT GTGGCCAGAT ACT-
GCGACCT CCCTAGCAAA CTGGGGCACA AACTTAATgg tggcggtgga tcaggcggag gtgggagtgg
aggtggaggg tctCCCGGGA TGGCGAGAAC CAAGCATCGC GTTACCAGGT CACAAC-
CTCG GAATCAAACT Ggtatcttaa atctgctttc tctttcaatt tttacttctg attttaccca gaattttagg ttttttattt

cgattttgtt aaccctagat ttcgaatctg aaatttgtag ATGCCGCCGG TGCTTCATCT TCTCAGGCGG
CAGGTCCAAC TACGgtacgg catctttttc cgtcttaggg tttccaatgt ttcttccttt tatcgttatg atcaaatttg tttatctatc
gaaattgaag ACCCCGACAA GGAGAGGCGG TGAAGGTGGA GATAATACTC AA-
CAAAgtga gttttttata tttgaagtct tttttttccc tcttttcatc tcttttgttt gtgaagttat tcttttgtaa catctgcagC AAATC-
CTACA ACTTCACCAG CTACTGGTAC AAGGgtaaga tttttgtgac cattgcttat gaactgcttc aactttgatt
tcgttattaa gctgacaaaa ttctcgtttt ggtttgtcaa gAGAGGGGCT AAGAGATCCA GACAGGCTAT GC-
CACGAGgt ttgttttaaa aaaaaaacca atctcttgtg atatccctga gaatacagga cacttagtgt gtttaaaact aatcttcggt
gttgtccttg tagGCTCACA GAAGAAGTCT TATCGATACA GGCCAGGAAC CGTTGCTCTA
AAAGAGATTC GCCATTTCCA GAAGCAGACA AACCTTCTTA TTCCGGCTGC CAGTTTC
ATA AGAGAAgtta gttactcttt ttcttaccag ccataataag tttcacagct taacaatatt catatatact aacagaggca
caagcctttt ggtgtttaat gtggctagtt ttaggatttg cacaccccac acatatctga gcatcaatgc agtgtacata gtgagtgata
tagcaattta actaaaattc agagtaatcg tgaggccaac cctccttgtt taaggagtgt gtaatctagt ttgtctttga ggttatgagc
tcatagattc agaaccatat gattcctgta gctacaaaac tcaacatgaa tcgtcagtga tgtggaaatg ctgatttgtg
ttacaaacaa actattttac attgtttttc cagGTGAGAA GTATAACCCA TATGTTGGCC CCTCCCCAAA
TCAATCGTTG GACAGCTGAA GCTCTTGTTG CTCTTCAAGA Ggtaccaatc cttcaacttt ttctttatac
gaatgtatga atatagatat agagatagtc acacatttca actaatgtca ttccccttga tgaccaatca acctaatcac
acaaattctt tgtggtagGC GGCAGAAGAT TACTTGGTTG GTTTGTTCTC AGATTCAATG CTCT-
GTGCTA TCCATGCAAG ACGTGTTACT CTAAgtaagt actctaaaag aagacatttt tcagtctcaa cttaggaatc
acaagcatac attttatatc cctttgaatc attagttact tgaatatcat atataaaatg cttatctata tctgtttttt gttcatatca
gTGAGAAAAG ACTTTGAACT TGCACGCCGG CTTGGAGGAA AAGGCAGACC ATG-
GTGAtag aaaactcact cactattcac atctcttaca ttgtaagtga ggatcgaata gttaacaaca agtgtgtcgt aggttgtata
aggttattct ctccttgtct cttggtgatg catgtccttt tattttacta aatgatctga ccaaaacctt cttttgaaac ataactggaa acccgaattt
ggtttgattc caatatctag gctgtaaaac ttctttctct atgtgtgttt ctatctgctt ttcgttggat ctgattgttt tccattgaga
ttccacttga caaaaacgtt tctttcgtta agttctaact tccaaaggtt catgtatata taatttgaag cctcagttag aaagggttgt
gattgaaaga agaaaaaaaa agtaagagga agatttgaaa gaacatgtgt tttgggaggg ctttagtaga aatatctctc agacgagtga
cgagtggatt cccaattaca acttgtcaag gcatattaaa aaggttgaga tatccttagc acttcaaatt taaataattc
atattgattt ttagtgataa ttcgcataaa acatttgtga tatcatagtg ttgtcactaa aaaaaacact ataaattttc gaaaagctcg
aatttcgaat ttggtgatat ttatttcatt gtcaactctt ttaagttgcc tctttttctt gggttctcta tagtctcttt tgttttgttt tttacatcct
ttatgatctc tctctgtaca ttcgtaaaaa ttgaatgcag accgaaaaaa ctaaacagaa acgaggatgg caaaagttgg atgcgcccat
gaatcatgca acacaatggt cccaagttcc caacagattc attcgcaaaa attcagagta gaccgaaaga aaaaacaaat
tgtagtatta taaaaaaaaa gaaagaaaga ggatgggtca agtgaaggc tcatgaatca tgcgatggtc ccaacaaatt aaatgaaata
taaaatccac gtgtcatgtt cagttgcaac tgcaAAGGGT GGGCGCGCCG ACCCAGCTTT CTTG-
TACAAA GTTGGCATTA TAAGAAAGCA TTGCTTATCA ATTTGTTGCA ACGAACAGGT
CACTATCAGT CAAAATAAAA TCATTATTTG CCATCCAGCT GATATCCCCT ATAGT-

GAGTC GTATTACATG GTCATAGCTG TTTCCTGGCA GCTCTGGCCC GTGTCTCAAA
ATCTCTGATG TTACATTGCA CAAGATAAAA ATATATCATC ATGAACAATA AAACT-
GTCTG CTTACATAAA CAGTAATACA AGGGGTGTTA TGAGCCATAT TCAACGGGAA
ACGTCGAGGC CGCGATTAAA TTCCAACATG GATGCTGATT TATATGGGTA TAAATGGG
CT CGCGATAATG TCGGGCAATC AGGTGCGACA ATCTATCGCT TGTATGGGAA GC-
CCGATGCG CCAGAGTTGT TTCTGAAACA TGGCAAAGGT AGCGTTGCCA ATGAT-
GTTAC AGATGAGATG GTCAGACTAA ACTGGCTGAC GGAATTTATG CCTCTTCCGA
CCATCAAGCA TTTTATCCGT ACTCCTGATG ATGCATGGTT ACTCACCACT GCGATC-
CCCG GAAAAACAGC ATTCCAGGTA TTAGAAGAAT ATCCTGATTC AGGTGAAAAT
ATTGTTGATG CGCTGGCAGT GTTCCTGCGC CGGTTGCATT CGATTCCTGT TTGTAATT
GT CCTTTTAACA GCGATCGCGT ATTTCGTCTC GCTCAGGCGC AATCACGAAT GAATA
ACGGT TTGGTTGATG CGAGTGATTT TGATGACGAG CGTAATGGCT GGCCTGTTGA
ACAAGTCTGG AAAGAAATGC ATAAACTTTT GCCATTCTCA CCGGATTCAG TCGT-
CACTCA TGGTGATTTC TCACTTGATA ACCTTATTTT TGACGAGGGG AAATTAATAG
GTTGTATTGA TGTTGGACGA GTCGGAATCG CAGACCGATA CCAGGATCTT GCCATC-
CTAT GGAACTGCCT CGGTGAGTTT TCTCCTTCAT TACAGAAACG GCTTTTTCAA
AAATATGGTA TTGATAATCC TGATATGAAT AAATTGCAGT TTCATTTGAT GCTCGAT-
GAG TTTTTCTAAT CAGAATTGGT TAATTGGTTG TAACACTGGC AGAGCATTAC GCT-
GACTTGA CGGGACGGCG CAAGCTCATG ACCAAAATCC CTTAACGTGA GTTACGCGTC
GTTCCACTGA GCGTCAGACC CCGTAGAAAA GATCAAAGGA TCTTCTTGAG ATC-
CTTTTTT TCTGCGCGTA ATCTGCTGCT TGCAAACAAA AAAACCACCG CTACCAGCGG
TGGTTTGTTT GCCGGATCAA GAGCTACCAA CTCTTTTTCC GAAGGTAACT GGCTTCAG
CA GAGCGCAGAT ACCAAATACT GTCCTTCTAG TGTAGCCGTA GTTAGGCCAC CACTT
CAAGA ACTCTGTAGC ACCGCCTACA TACCTCGCTC TGCTAATCCT GTTACCAGTG
GCTGCTGCCA GTGGCGATAA GTCGTGTCTT ACCGGGTTGG ACTCAAGACG ATAGT-
TACCG GATAAGGCGC AGCGGTCGGG CTGAACGGGG GGTTCGTGCA CACAGCCCAG
CTTGGAGCGA ACGACCTACA CCGAACTGAG ATACCTACAG CGTGAGCATT GAGAAA
GCGC CACGCTTCCC GAAGGGAGAA AGGCGGACAG GTATCCGGTA AGCGGCAGGG
TCGGAACAGG AGAGCGCACG AGGGAGCTTC CAGGGGGAAA CGCCTGGTAT CTT-
TATAGTC CTGTCGGGTT TCGCCACCTC TGACTTGAGC GTCGATTTTT GTGATGCTCG
TCAGGGGGGC GGAGCCTATG GAAAAACGCC AGCAACGCGG CCTTTTTACG GTTC-
CTGGCC TTTTGCTGGC CTTTTGCTCA CATGTT

Destination vector
R4pGWB501
tttcacgccc ttttaaatat ccgattattc taataaacgc tcttttctct taggtttacc cgccaatata tcctgtcaaa cactgatagt
ttaaactgaa ggcgggaaac gacaatctga tccaagctca agctgctcta gcattcgcca ttcaggctgc gcaactgttg ggaagggc
ga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagcttgtgg atcccccatc acaactttgt atagaaaagt tgaacgagaa
acgtaaaatg atataaatat caatatatta aattagattt tgcataaaaa acagactaca taatactgta aaacacaaca
tatccagtca ctatggcggc cacatttaaa tgtcgagggg ccgcattagg caccccaggc tttacacttt atgcttccgg ctcgtataat
gtgtggattt tgagttagga tccgtcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac
caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct
ggatattacg gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc
tcatccggaa ttccgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac
tgaaacgttt tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga
aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa
cgtggccaat atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat
tcaggttcat catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca acagtactgc gatgagtggc
agggcggggc gtaaagatct ggatccggct tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt ataagaatat
atactgatat gtatacccga agtatgtcaa aaagaggtgt gctatgaagc agcgtattac agtgacagtt gacagcgaca
gctatcagtt gctcaaggca tatatgatgt caatatctcc ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga
acgctggaaa gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa cggctctttt gctgacgaga
acaggggctg gtgaaatgca gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt
gacacgcccg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc
ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtctccgtt atcggggaag aagtg-

gctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac
acagccagtc tgcaggtcga ccatagtgac tggatatgtt gtgttttaca gtattatgta gtctgttttt tatgcaaaat ctaatttaat
atattgatat ttatatcatt ttacgtttct cgttcagctt tcttgtacaa agtggttgat aacagcgctt agagctcgaa tttccccgat
cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg tcttgcgatg attatcatat aatttctgtt gaattacgtt
aagcatgtaa taattaacat gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat ttaatacgcg
atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt gtcatctatg ttactagatc gggaattggt tccggaacca
attcgtaatc atgtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaggc tgaattaggc gcgcctattt ctattaattc ccgatctagt
aacatagatg acaccgcgcg cgataattta tcctagtttg cgcgctatat tttgttttct atcgcgtatt aaatgtataa ttgcgggact
ctaatcataa aaacccatct cataaataac gtcatgcatt acatgttaat tattacatgc ttaacgtaat tcaacagaaa ttatatgata
atcatcgcaa gaccggcaac aggattcaat cttaagaaac tttattgcca aatgtttgaa cgatcgggga aattcggggg atcgatcccg
cgaactgtgg acgagaactg tgccaccaag cgtaaggccg ttctctcgca tttgccttgc taggctcgcg cgagttgctg
gctgaggcgt tctcgaaatc agctcttgtt cggtcggcat ctactctatt cctttgccct cggacgagtg ctggggcgtc ggtttccact
atcggcgagt acttctacac agccatcggt ccagacggcc gcgcttctgc gggcgatttg tgtacgcccg acagtcccgg ctccggatcg
gacgattgcg tcgcatcgac cctgcgccca agctgcatca tcgaaattgc cgtcaaccaa gctctgatag agttggtcaa
gaccaatgcg gagcatatac gcccggagcc gcggcgatcc tgcaagctcc ggatgcctcc gctcgaagta gcgcgtctgc
tgctccatac aagccaacca cggcctccag aagaagatgt tggcgacctc gtattgggaa tccccgaaca tcgcctcgct ccagtcaatg
accgctgtta tgcggccatt gtccgtcagg acattgttgg agccgaaatc cgcgtgcacg aggtgccgga cttcggggca
gtcctcggcc caaagcatca gctcatcgag agcctgcgcg acggacgcac tgacggtgtc gtccatcaca gtttgccagt gatacacatg
gggatcagca atcgcgcata tgaaatcacg ccatgtagtg tattgaccga ttccttgcgg tccgaatggg ccgaacccgc
tcgtctggct aagatcggcc gcagcgatcg catccatggc ctccgcgacc ggctgcagaa cagcgggcag ttcggtttca
ggcaggtctt gcaacgtgac accctgtgca cggcgggaga tgcaataggt caggctctcg ctgaatgccc caatgtcaag cacttccgga
atcgggagcg cggccgatgc aaagtgccga taaacataac gatctttgta gaaaccatcg gcgcagctat ttacccgcag
gacatatcca cgccctccta catcgaagct gaaagcacgagattcttcgc cctccgagag ctgcatcagg tcggagacgc tgtcgaactt
ttcgatcaga aacttctcga cagacgtcgc ggtgagttca ggctttttca tatcttattg ccccccggga tcagatctgg
attgagagtg aatatgagac tctaattgga taccgagggg aatttatgga acgtcagtgg agcatttttg acaagaaata tttgctagct
gatagtgacc ttaggcgact tttgaacgcg caataatggt ttctgacgta tgtgcttagc tcattaaact ccagaaaccc gcggctgagt
ggctccttca acgttgcggt tctgtcagtt ccaaacgtaa aacggcttgt cccgcgtcat cggcgggggt cataacgtga
ctcccttaat tctccgctca tgatcttgat cccctgcgcc atcagatcct tggcggcaag aaagccatcc agtttacttt gcagggcttc
ccaaccttac cagagggcgc cccagctggc aattccggtt cgcttgctgt ccataaaacc gcccagtcta gctatcgcca tgtaagccca
ctgcaagcta cctgctttct ctttgcgctt gcgttttccc ttgtccagat agcccagtag ctgacattca tccggggtca
gcaccgtttc tgcggactgg ctttctacgt gttccgcttc ctttagcagc ccttgcgccc tgagtgcttg cggcagcgtg aagctgatcc
gtcgagatta atagaaataa attcagccta attcggcgtt aattcagtac attaaaaacg tccgcaatgt gttattaagt
tgtctaagcg tcaatttgtt tacaccacaa tatatcctgc caccagccag ccaacagctc cccgaccggc agctcggcac aaaat-

cacca ctcgatacag gcagcccatc agtccgggac ggcgtcagcg ggagagccgt tgtaaggcgg cagactttgc tcatgttacc
gatgctattc ggaagaacgg caactaagct gccgggtttg aaacacggat gatctcgcgg agggtagcat gttgattgta acgatgacag
agcgttgctg cctgtgatca attcgggcac gaacccagtg gacataagcc tgttcggttc gtaagctgta atgcaagtag
cgtatgcgct cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg taacggcgca gtggcggttt tcatggcttg ttatgactgt
ttttttgggg tacagtctat gcctcgggca tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga tgttatggag
cagcaacgat gttacgcagc agggcagtcg ccctaaaaca aagttaaaca tcatggggga agcggtgatc gccgaagtat
cgactcaact atcagaggta gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg ccgtacattt gtacggctcc gcagtggatg
gcggcctgaa gccacacagt gatattgatt tgctggttac ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg
atcaacgacc ttttggaaac ttcggcttcc cctggagaga gcgagattct ccgcgctgta gaagtcacca ttgttgtgca cgacgacatc
attccgtggc gttatccagc taagcgcgaa ctgcaatttg gagaatggca gcgcaatgac attcttgcag gtatcttcga
gccagccacg atcgacattg atctggctat cttgctgaca aaagcaagag aacatagcgt tgccttggta ggtccagcgg cggaggaact
ctttgatccg gttcctgaac aggatctatt tgaggcgcta aatgaaacct taacgctatg gaactcgccg cccgactggg
ctggcgatga gcgaaatgta gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg
actgggcaat ggagcgcctg ccggcccagt atcagcccgt catacttgaa gctagacagg cttatcttgg acaagaagaa
gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg tccactacgt gaaaggcgag atcaccaagg tagtcggcaa ataatgtcta
gctagaaatt cgttcaagcc gacgccgctt cgcggcgcgg cttaactcaa gcgttagatg cactaagcac ataattgctc
acagccaaac tatcaggtca agtctgcttt tattattttt aagcgtgcat aataagccct acacaaattg ggagatatat catgcatgac
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
aaacgcgcga ggcagggtgc cttgatgtgg gcgccggcgg tcgagtggcg acggcgcggc ttgtccgcgc cctggtagat
tgcctggccg taggccagcc atttttgagc ggccagcggc cgcgataggc cgacgcgaag cggcggggcg tagggagcgc
agcgaccgaa gggtaggcgc tttttgcagc tcttcggctg tgcgctggcc agacagttat gcacaggcca ggcgggtttt aagagtttta
ataagtttta aagagtttta ggcggaaaaa tcgccttttt tctcttttat atcagtcact tacatgtgtg accggttccc aatg-

tacggc tttgggttcc caatgtacgg gttccggttc ccaatgtacg gctttgggtt cccaatgtac gtgctatcca caggaaagag
accttttcga cctttttccc ctgctagggc aatttgccct agcatctgct ccgtacatta ggaaccggcg gatgcttcgc cctcgatcag
gttgcggtag cgcatgacta ggatcgggcc agcctgcccc gcctcctcct tcaaatcgta ctccggcagg tcatttgacc
cgatcagctt gcgcacggtg aaacagaact tcttgaactc tccggcgctg ccactgcgtt cgtagatcgt cttgaacaac catctggctt
ctgccttgcc tgcggcgcgg cgtgccaggc ggtagagaaa acggccgatg ccgggatcga tcaaaaagta atcggggtga
accgtcagca cgtccgggtt cttgccttct gtgatctcgc ggtacatcca atcagctagc tcgatctcga tgtactccgg ccgcccggtt
tcgctcttta cgatcttgta gcggctaatc aaggcttcac cctcggatac cgtcaccagg cggccgttct tggccttctt cgtacgctgc
atggcaacgt gcgtggtgtt taaccgaatg caggtttcta ccaggtcgtc tttctgcttt ccgccatcgg ctcgccggca
gaacttgagt acgtccgcaa cgtgtggacg gaacacgcgg ccgggcttgt ctcccttccc ttcccggtat cggttcatgg attcggttag
atgggaaacc gccatcagta ccaggtcgta atcccacaca ctggccatgc cggccggccc tgcggaaacc tctacgtgcc
cgtctggaag ctcgtagcgg atcacctcgc cagctcgtcg gtcacgcttc gacagacgga aaacggccac gtccatgatg ctgcgactat
cgcgggtgcc cacgtcatag agcatcggaa cgaaaaaatc tggttgctcg tcgcccttgg gcggcttcct aatcgacggc
gcaccggctg ccggcggttg ccgggattct ttgcggattc gatcagcggc cgcttgccac gattcaccgg ggcgtgcttc tgcctcgatg
cgttgccgct gggcggcctg cgcggccttc aacttctcca ccaggtcatc acccagcgcc gcgccgattt gtaccgggcc
ggatggtttg cgaccgctca cgccgattcc tcgggcttgg gggttccagt gccattgcag ggccggcaga caacccagcc gcttacgcct
ggccaaccgc ccgttcctcc acacatgggg cattccacgg cgtcggtgcc tggttgttct tgattttcca tgccgcctcc
tttagccgct aaaattcatc tactcattta ttcatttgct catttactct ggtagctgcg cgatgtattc agatagcagc tcggtaatgg
tcttgccttg gcgtaccgcg tacatcttca gcttggtgtg atcctccgcc ggcaactgaa agttgacccg cttcatggct ggcgtgtctg
ccaggctggc caacgttgca gccttgctgc tgcgtgcgct cggacggccg gcacttagcg tgtttgtgct tttgctcatt
ttctctttac ctcattaact caaatgagtt ttgatttaat ttcagcggcc agcgcctgga cctcgcgggc agcgtcgccc tcgggttctg
attcaagaac ggttgtgccg gcggcggcag tgcctgggta gctcacgcgc tgcgtgatac gggactcaag aatgggcagc
tcgtacccgg ccagcgcctc ggcaacctca ccgccgatgc gcgtgccttt gatcgcccgc gacacgacaa aggccgcttg tagccttcca
tccgtgacct caatgcgctg cttaaccagc tccaccaggt cggcggtggc ccatatgtcg taagggcttg gctgcaccgg
aatcagcacg aagtcggctg ccttgatcgc ggacacagcc aagtccgccg cctggggcgc tccgtcgatc actacgaagt cgcgccggcc
gatggccttc acgtcgcggt caatcgtcgg gcggtcgatg ccgacaacgg ttagcggttg atcttcccgc acggccgccc
aatcgcgggc actgccctgg ggatcggaat cgactaacag aacatcggcc ccggcgagtt gcagggcgcg ggctagatgg
gttgcgatgg tcgtcttgcc tgacccgcct ttctggttaa gtacagcgat aaccttcatg cgttcccctt gcgtatttgt ttatttactc
atcgcatcat atacgcagcg accgcatgac gcaagctgtt ttactcaaat acacatcacc tttttagacg gcggcgctcg gtttcttcag
cggccaagct ggccggccag gccgccagct tggcatcaga caaaccggcc aggatttcat gcagccgcac ggttgagacg
tgcgcgggcg gctcgaacac gtacccggcc gcgatcatct ccgcctcgat ctcttcggta atgaaaaacg gttcgtcctg gccgtcctgg
tgcggtttca tgcttgttcc tcttggcgtt cattctcggc ggccgccagg gcgtcggcct cggtcaatgc gtcctcacgg
aaggcaccgc gccgcctggc ctcggtgggc gtcacttcct cgctgcgctc aagtgcgcgg tacagggtcg agcgatgcac
gccaagcagt gcagccgcct ctttcacggt gcggccttcc tggtcgatca gctcgcgggc gtgcgcgatc tgtgccgggg
tgagggtagg gcgggggcca aacttcacgc ctcgggcctt ggcggcctcg cgcccgctcc gggtgcggtc gatgattagg

gaacgctcga actcggcaat gccggcgaac acggtcaaca ccatgcggcc ggccggcgtg gtggtgtcgg cccacggctc
tgccaggcta cgcaggcccg cgccggcctc ctggatgcgc tcggcaatgt ccagtaggtc gcgggtgctg cgggccaggc
ggtctagcct ggtcactgtc acaacgtcgc cagggcgtag gtggtcaagc atcctggcca gctccgggcg gtcgcgcctg
gtgccggtga tcttctcgga aaacagcttg gtgcagccgg ccgcgtgcag ttcggcccgt tggttggtca agtcctggtc gtcggtgctg
acgcgggcat agcccagcag gccagcggcg gcgctcttgt tcatggcgta atgtctccgg ttctagtcgc aagtattcta
ctttatgcga ctaaaacacg cgacaagaaa acgccaggaa aagggcaggg cggcagcctg tcgcgtaact taggacttgt gcgacatgtc
gttttcagaa gacggctgca ctgaacgtca gaagccgact gcactatagc agcggagggg ttggatcaaa gtactttgat
cccgagggga accctgtggt tggcatgcac atacaaatgg acgaacggat aaacct
pDONR P4-P1r

pDONR221