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Nicole Sleiman
Cell Biology
Cyclin TTHERM_00693080 (CYC13):
Abstract:
Tetrahymena’s sexual cycle is called conjugation. This cycle is one unique process where
two cells of different mating types are put under stressful conditions, and then pair to
exchange haploid gamete nuclei. Conjugation results in two daughter cells with one silent
micronucleus and a transcriptionally active macronucleus. To drive the cell cycle in its
different stages, cyclins were found to have an important role. Expression data from
public database on the Cyclin gene TTHERM_0069380 (CYC13) was collected in order
to determine whether or not it is involved in the regulation of the different steps of the
conjugation of Tetrahymena thermophila. RT_PCR analysis was then completed to
complement these data. During conjugation this gene was expressed at specific times.
After collecting the data from this study and comparing it to other studies that have
specified details of the expression of this gene during 18 hour conjugation, a conclusion
was deducted about the specific role of this cyclin gene in the different steps of the
conjugation cycle. This gene was found to be expressed between hour 4 and 9.
Introduction:
In Tetrahymena’s life cycle, the sexual division process where two cells pair to exchange
haploid gamete nuclei is called conjugation. Conjugation occurs when two cells of
different mating type (I-VII) undergoes stressful conditions. This nuclear event includes
meiosis, gamete nucleus formation, fertilization and nuclear differentiation.
Microuclei in the paired cells undergo meiosis, and by doing so they generate four
haploid pronuclei. Only one of these pronuclei is kept and the other three are destroyed.
These remaining pronuclei divides and form two gametic nuclei called migratory and
stationary pronucleus. The migratory pronuclei are exchanged in the two cells; they then
form a zygotic nucleus in each cell after they are fused with a stationary pronucleus. The
zygotic nucleus is then divided twice forming four identical nuclei where two of them in
each cell become an immature macronucleus “anlagen” that will eventually undergo
genome rearrangements such as chromosome breakage, programmed DNA elimination,
and telomere addition, to complete the macronuclear genome. Some clones degrade
others divides mitotically to form two daughter cells with one silent micronucleus and a
transcriptionally active macronucleus (Miao, et al 2009). Fig 1 shows in more details
what happens during the conjugation process.
Fig 1. Events in the Tetrahymena thermophila conjugation cycle. Taken from Miao, et
al., PLoS ONE. 2009; 4(2): e4429 (Miao et al, 2009)
Cyclins are proteins that can control the progression of cells through the cell life cycle.
Cyclins concentration varies at specific stages, some are produced and others are
degraded as needed in order to drive the cell through the different stages. Cyclindependent kinases (CDKs) become activated when cyclins bind to them. Once CDKs
become activated, they activate or deactivate via phosphorilation of proteins needed for
specific stages in the cell cycle.As Cyclins are degraded, others become expressed to
direct the cell to move on to the next stage (Zhang et al. 1999) .
Specific cyclin genes were researched to study their importance throughout the
conjugation cycle.
TTHERM_00693080 gene was found in the blast search, and it was found to be a cyclin
gene. This gene was tested further to study its importance in division cycles.
Method:
Cyclin genes were identified at the Tetrahymena Genome Database
(www.ciliate.org) by searching for proteins with the keyword “cyclin”. A BLAST search
with a cyclin protein sequence ensured that all cyclin genes were identified using this
method. Microarray data during conjugation (Miao, et al., PLoS ONE. 2009; 4(2):
e4429) were collected for each gene from the Tetrahymena Gene Expression Database
(TGED; http://tged.ihb.ac.cn). PCR primers flanking an intron were generated for each
gene using Primer3 (Steve Rozen and Helen J. Skaletsky 2000) and ordered from
Integrated DNA Technology (Coralville, IA). The forward primer
TTHERM_00693080cDNA-F was (5’-CACCGTTATATTTTATTTATAAAAGTAAGT-3’),
and the reverse primer was (5-TCAAATATTGGTTAGTTGTTAAG-3’) .Oligo-dT-
primed M-MLV reverse transcription (RT; Ambion) was performed on RNA collected
from control cells and from cells at various stages of conjugation using the Trizol reagent
(Invitrogen) according to the manufacturer’s protocol. 1 mL of cells (2.1 x 103 cells/mL)
was collected at each time point, pelleted at 6k rpm, supernatant discarded, and cells
resuspended in 1 mL of Trizol. 180 ng of each template RNA was used per reverse
transcription reaction. cDNA was diluted 1:5 and used as a template for PCR (How
much RNA for RT? ) PCR was performed in 25 uL reactions using GOTaq (Fisher,
Hampton, NH) with 1 uL of each primer (10 uM). 15 uL of completed PCR reaction
products were separated on a 2% agarose gel. DNA bands were visualized using
ethidium bromide and photographed with a Kodak EDAS290 imaging system. Band
intensities were determined using ImageJ (Abramoff, M.D., Magelhaes, P.J., Ram, S.J.
"Image Processing with ImageJ". Biophotonics International, volume 11, issue 7, pp. 3642, 2004).
Result:
RT-PCR analysis showed a peak at C7 and another around C9.5, these results were
slightly different from those found in the microarray graph where the peaks were located
at points C6 and C16.
Fig 2. Note: For growing cells, L-l, L-m and L-h correspond respectively to ~1105 cells/ml,
~3.5105 cells/ml and ~1106 cells/ml. For starvation, ~2105 cells/ml were collected at 0, 3, 6, 9, 12,
15 and 24 hours（referred to as S-0, S-3, S-6, S-9, S-12, S-15 and S-24). For conjugation, equal
volumes of B2086 and CU428 cells were mixed, and samples were collected at 0, 2, 4, 6, 8, 10, 12, 14,
16 and 18 hours after mixing (referred to as C-0, C-2, C-4, C-6, C-8, C-10, C-12, C-14, C-16 and C18). http://tged.ihb.ac.cn/search.aspx?keyword=TTHERM_00693080
60
Signal Intensity
50
40
30
20
10
7
6
5
4
3
2
1
0
8
C1
C1
C1
C1
C1
C1
C1
C1
C9
C1
C8
C7
C6
C5
C4
C3
C2
C1
C0
42
7V
42
8V
42
7S
42
8S
0
RNA Collection Time Points
Fig 3. RT_PCR data was collected for two different mating cell types during vegetative,
starvation, and through 18 hours of conjugation after being mixed.
427V 428V 427
428V
S
428S C0
C1
C2
C3
C4
C5
C6
C7
C8
C9
cjs C18
C10 C11 C12 C13 C14 C15 C16 C17
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sga
ugf
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gfja
g
Fig 4. Gel electrophoresis of Mrna during vegetative, starvation, and through 18 hours of
conjugation after being mixed.
Discussion:
The cyclin gene TTHERM_00693080 (CYC13) studied in this experiment by RT_PCR
generallly matched the data found in the public database except for a few signals that
were not expressed as much as they should have been expressed. This cyclin gene was
not expressed during starvation periods. Once the cells were mixed together, this gene’s
expression started increasing; this gene had its peak around time C5. This gene
expression started decreasing after time C7. The data collected in this experiment
exhibited the same trend as the data from the microarray analysis. Both graphs have a big
peak, followed with a smaller peak. The difference between those two graphs is the time
at which the peaks are. The microarray graph data was taken every two hours and that
may explain some of the discrepancies found between the time points.
Between time C3 and C7, meosis 1 and 2 happen. From the four haploid cells that form,
one meotic product undergoes mitosis; an exchange of pronuclei occurs followed by
fertilization, and by the1st and 2nd postgygotic divisions.
During the time this gene is expressed, microuclei in the paired cells undergo meiosis,
and by doing so they generate four haploid pronuclei. Only one of these pronuclei is kept
and the other three are destroyed. These remaining pronuclei divides and form two
gametic nuclei called migratory and stationary pronucleus. The migratory pronuclei are
exchanged in the two cells; they then form a zygotic nucleus in each cell after they are
fused with a stationary pronucleus. The zygotic nucleus is then divided twice forming
four identical nuclei.
All these steps have to do with the micronucleus, and therefore it can be assumed that this
cyclin gene makes sure that the micronuclei undergo proper cell division during
conjugation, and it promotes the development of the new macroneuclei when its
expression starts decreasing. It may also function in helping to pair micronuclei in
preparing them for division. Another possibility that may serve as a function for this
cyclin is its involvement in formation of cellular structures that aid in the transportation
of the haploid cells from one cell to another which would lead to their fusion.
References:
Miao, et al., Microarray Analyses of Gene Expression during the Tetrahymena
thermophila Life Cycle. PloSONE. 2009; 4(2): e4429.
Zhang H, Adl SM, Berger JD. 1999. Two distinct classes of mitotic cyclin homologues,
Cyc1 and Cyc2, are involved in cell cycle regulation in the ciliate Paramecium
tetraurelia. Journal of Eukaryotic Microbiology. 46(6):585-96.