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Identifying the function of GLYMA10, a potential new APC activator in
(Glycine max L. Merr)
David Grimm and Nicole Dafoe
Department of Biology, Slippery Rock University, Slippery Rock, PA 16057, USA
Materials and Methods
Introduction
C-box
A typical cell cycle is characterized by a growth phase followed by
DNA replication and then finally cell division. This process generates
genetically identical cells. Alternatively, in the process known as
endoreduplication, cells grow, and DNA replication occurs, but the
cell does not divide. In eukaryotes and specifically plants, CDC20 and
CCS52 proteins control when a cell undergoes either mitosis or
endoreduplication respectively through activating the anaphase
promoting complex (APC). There have been a variety of CDC20 and
CCS52 genes discovered, yet more information of these cell cycle
regulators needs to be researched. What is currently known is that
the APC is activated through targeting specific proteins called cyclins,
which control the progression of a cell cycle, for degradation. This
research project focuses on a potential third type of APC activator in
soybean (Glycine max), Glyma.10G117000.1 (GLYMA10). GLYMA10
has been identified in the genome of soybean yet no function is
currently known. Within the soybean genome there are 4 CDC20
genes and 6 CCS52 genes. GLYMA10 shares sequence similarities
with CDC20 and CCS52 proteins (Figure 1). Preliminary data shows
that like CDC20 and CCS52 proteins, GLYMA10 has defining traits
such as a conserved C-box, WD40 repeats, RVL motif and an IR tail
which are all imperative to APC activating proteins (Figure 2). Cell
cycle regulators need a conserved C- box and IR tail to bind to the
APC. The RVL motif found within the WD40 domain is involved with
cyclin binding. In Arabidopsis thaliana, there are 4 CDC20 genes and
3 CCS52 genes, but there is no gene similar to GLYMA10 present
indicating that GLYMA10 is a possible new, unique class of APC
activator (Figure 1).
To further understand GLYMA10 and its function, GLYMA10 will be
expressed in Schizosaccharomyces pombe, a model system to study
cell division, and the phenotype will be examined to determine if
overexpressing this gene results in any unique yeast phenotypes
related to cell division.
RNA extraction from soybean tissue✓
↓
Convert RNA to cDNA using reverse transcriptase✓
↓
Amplify GLYMA10 using PCR ✓
↓
Clone GLYMA10 genes into expression vector
↓
Transform S. pombe with expression vectors
↓
Analyze phenotypes
Results
• Based on sequence comparisons, it appears that GLYMA10 may be
a new type of APC activator
• RNA has been extracted from various soybean tissues
• The RNA has been converted to cDNA
• cDNA samples have been used for PCR
• Full-length GLYMA10 was amplified from apical meristem tissue, a
tissue where cell division occurs
• We have tried cloning GLYMA10 into two different yeast expression
vectors (pSLF102 and pREP4X), but have yet to be successful
Future Research
RVL Motif
• Transform S. pombe with GLYMA10 gene
• Overexpress GLYMA10 genes in soybean to see where the gene is
expressed
• Use qPCR to study expression patterns of GLYMA10 in soybeans beyond
the apical meristem
• Bioinformatics data on the soybean genome suggests that
GLYMA10 could be concentrated in flowers of soybean
References
IR Tail
Figure 1. Soybean and Arabidopsis both share similar APC
activator genes except the novel GLYMA10. Phylogenetic tree of
APC activator genes Glycine max (Gm) and Arabidopsis thaliana
(At).
Expression of soybean GLYMA10 genes in S. pombe
Figure 2. Alignment of GLYMA10 with CDC20 proteins. Sequences were
retrieved from the Joint Genome Institute database with the following
accession numbers: GLYMA10 (Glyma.10G117000.1), CDC20.1
(Glyma.11G012400.1), CDC20.2 (Glyma.01G229600.1), CDC20.3
(Glyma.03G204700.1), and CDC20.4 (Glyma.08G228100.1). Highlighted
letters represent conserved amino acids. Highlighted amino acids are
conserved.
Castro A, Bernis C, Vigneron S, Labbe JC, Lorca T (2005) The anaphase-promoting complex: a key factor in the regulation of cell
cycle. Oncogene 24 (3):314-325. doi:10.1038/sj.onc.1207973
Dafoe NJ, Huffaker A, Vaughan MM, Duehl AJ, Teal PE, Schmelz EA (2011) Rapidly Induced Chemical Defenses in Maize Stems
and Their Effects on Short-term Growth of Ostrinia nubilalis. Journal of Chemical Ecology 37 (9):984-991.
doi:10.1007/s10886-011-0002-9
Fulop K, Tarayre S, Kelemen Z, Horvath G, Kevei Z, Nikovics K, Bako L, Brown S, Kondorosi A, Kondorosi E (2005) Arabidopsis
anaphase-promoting complexes - Multiple activators and wide range of substrates might keep APC perpetually busy. Cell
Cycle 4 (8):1084-1092
Heyman J, De Veylder L (2012) The Anaphase-Promoting Complex/Cyclosome in Control of Plant Development. Molecular Plant
5 (6):1182-1194. doi:10.1093/mp/sss094
Kevei Z, Baloban M, Da Ines O, Tiricz H, Kroll A, Regulski K, Mergaert P, Kondorosi E (2011) Conserved CDC20 Cell Cycle Functions
Are Carried out by Two of the Five Isoforms in Arabidopsis thaliana. Plos One 6 (6). doi:10.1371/journal.pone.0020618
Lima MD, Eloy NB, Pegoraro C, Sagit R, Rojas C, Bretz T, Vargas L, Elofsson A, de Oliveira AC, Hemerly AS, Ferreira PCG (2010)
Genomic evolution and complexity of the Anaphase-promoting Complex (APC) in land plants. Bmc Plant Biology 10.
doi:10.1186/1471-2229-10-254
Tarayre S, Vinardell JM, Cebolla A, Kondorosi A, Kondorosi E (2004) Two classes of the Cdh1-type activators of the anaphasepromoting complex in plants: Novel functional domains and distinct regulation. Plant Cell 16 (2):422-434.
doi:10.1105/tpc.018952
Acknowledgements
We would like to thank the Department of Biology at Slippery Rock University for
their wonderful support and the Faculty/ Student Research Grant for funding this
research.
(Glycine max L. Merr)
David Grimm and Nicole Dafoe
Department of Biology, Slippery Rock University, Slippery Rock, PA 16057, USA
Materials and Methods
Introduction
C-box
A typical cell cycle is characterized by a growth phase followed by
DNA replication and then finally cell division. This process generates
genetically identical cells. Alternatively, in the process known as
endoreduplication, cells grow, and DNA replication occurs, but the
cell does not divide. In eukaryotes and specifically plants, CDC20 and
CCS52 proteins control when a cell undergoes either mitosis or
endoreduplication respectively through activating the anaphase
promoting complex (APC). There have been a variety of CDC20 and
CCS52 genes discovered, yet more information of these cell cycle
regulators needs to be researched. What is currently known is that
the APC is activated through targeting specific proteins called cyclins,
which control the progression of a cell cycle, for degradation. This
research project focuses on a potential third type of APC activator in
soybean (Glycine max), Glyma.10G117000.1 (GLYMA10). GLYMA10
has been identified in the genome of soybean yet no function is
currently known. Within the soybean genome there are 4 CDC20
genes and 6 CCS52 genes. GLYMA10 shares sequence similarities
with CDC20 and CCS52 proteins (Figure 1). Preliminary data shows
that like CDC20 and CCS52 proteins, GLYMA10 has defining traits
such as a conserved C-box, WD40 repeats, RVL motif and an IR tail
which are all imperative to APC activating proteins (Figure 2). Cell
cycle regulators need a conserved C- box and IR tail to bind to the
APC. The RVL motif found within the WD40 domain is involved with
cyclin binding. In Arabidopsis thaliana, there are 4 CDC20 genes and
3 CCS52 genes, but there is no gene similar to GLYMA10 present
indicating that GLYMA10 is a possible new, unique class of APC
activator (Figure 1).
To further understand GLYMA10 and its function, GLYMA10 will be
expressed in Schizosaccharomyces pombe, a model system to study
cell division, and the phenotype will be examined to determine if
overexpressing this gene results in any unique yeast phenotypes
related to cell division.
RNA extraction from soybean tissue✓
↓
Convert RNA to cDNA using reverse transcriptase✓
↓
Amplify GLYMA10 using PCR ✓
↓
Clone GLYMA10 genes into expression vector
↓
Transform S. pombe with expression vectors
↓
Analyze phenotypes
Results
• Based on sequence comparisons, it appears that GLYMA10 may be
a new type of APC activator
• RNA has been extracted from various soybean tissues
• The RNA has been converted to cDNA
• cDNA samples have been used for PCR
• Full-length GLYMA10 was amplified from apical meristem tissue, a
tissue where cell division occurs
• We have tried cloning GLYMA10 into two different yeast expression
vectors (pSLF102 and pREP4X), but have yet to be successful
Future Research
RVL Motif
• Transform S. pombe with GLYMA10 gene
• Overexpress GLYMA10 genes in soybean to see where the gene is
expressed
• Use qPCR to study expression patterns of GLYMA10 in soybeans beyond
the apical meristem
• Bioinformatics data on the soybean genome suggests that
GLYMA10 could be concentrated in flowers of soybean
References
IR Tail
Figure 1. Soybean and Arabidopsis both share similar APC
activator genes except the novel GLYMA10. Phylogenetic tree of
APC activator genes Glycine max (Gm) and Arabidopsis thaliana
(At).
Expression of soybean GLYMA10 genes in S. pombe
Figure 2. Alignment of GLYMA10 with CDC20 proteins. Sequences were
retrieved from the Joint Genome Institute database with the following
accession numbers: GLYMA10 (Glyma.10G117000.1), CDC20.1
(Glyma.11G012400.1), CDC20.2 (Glyma.01G229600.1), CDC20.3
(Glyma.03G204700.1), and CDC20.4 (Glyma.08G228100.1). Highlighted
letters represent conserved amino acids. Highlighted amino acids are
conserved.
Castro A, Bernis C, Vigneron S, Labbe JC, Lorca T (2005) The anaphase-promoting complex: a key factor in the regulation of cell
cycle. Oncogene 24 (3):314-325. doi:10.1038/sj.onc.1207973
Dafoe NJ, Huffaker A, Vaughan MM, Duehl AJ, Teal PE, Schmelz EA (2011) Rapidly Induced Chemical Defenses in Maize Stems
and Their Effects on Short-term Growth of Ostrinia nubilalis. Journal of Chemical Ecology 37 (9):984-991.
doi:10.1007/s10886-011-0002-9
Fulop K, Tarayre S, Kelemen Z, Horvath G, Kevei Z, Nikovics K, Bako L, Brown S, Kondorosi A, Kondorosi E (2005) Arabidopsis
anaphase-promoting complexes - Multiple activators and wide range of substrates might keep APC perpetually busy. Cell
Cycle 4 (8):1084-1092
Heyman J, De Veylder L (2012) The Anaphase-Promoting Complex/Cyclosome in Control of Plant Development. Molecular Plant
5 (6):1182-1194. doi:10.1093/mp/sss094
Kevei Z, Baloban M, Da Ines O, Tiricz H, Kroll A, Regulski K, Mergaert P, Kondorosi E (2011) Conserved CDC20 Cell Cycle Functions
Are Carried out by Two of the Five Isoforms in Arabidopsis thaliana. Plos One 6 (6). doi:10.1371/journal.pone.0020618
Lima MD, Eloy NB, Pegoraro C, Sagit R, Rojas C, Bretz T, Vargas L, Elofsson A, de Oliveira AC, Hemerly AS, Ferreira PCG (2010)
Genomic evolution and complexity of the Anaphase-promoting Complex (APC) in land plants. Bmc Plant Biology 10.
doi:10.1186/1471-2229-10-254
Tarayre S, Vinardell JM, Cebolla A, Kondorosi A, Kondorosi E (2004) Two classes of the Cdh1-type activators of the anaphasepromoting complex in plants: Novel functional domains and distinct regulation. Plant Cell 16 (2):422-434.
doi:10.1105/tpc.018952
Acknowledgements
We would like to thank the Department of Biology at Slippery Rock University for
their wonderful support and the Faculty/ Student Research Grant for funding this
research.