Jody Mallicoat
Gene Expression Profiling of Cyc1 in Tetrahymena thermophila
Abstract
Cyc1 is a putative cyclin responsible for regulation of the T. thermophila the sexual
cycle, conjugation. By running RT-PCR of mRNA collected during the T. thermophila cell
cycle and comparing the results to the TGED expression profile, the putative function of Cyc1
can be predicted. The RT-PCR results reveal moderate increases in expression of CYC1 at hours
1 and 7 of conjugation. CYC1 expression is particularly high during hours 13 through 16 of
conjugation. The TGED expression profile indicates that CYC1 expression builds up in starved
cells, drops at the beginning of conjugation (hour 0), and increases during hours 1-4, 6-8, and 1014 with the highest expression occurring during hours 11-14 of conjugation. Based on the results
of RT-PCR, the TGED expression profile, and known function of the homolog in S. cerevisiae, it
is reasonable to infer that Cyc1 functions in the regulation of DNA replication and/or spindle
formation. Further experimentation, including the localization of Cyc1 within the cell, is
necessary to make more educated predictions of function.
Introduction
Tetrahymena thermophila undergoes a sexual cell cycle under starvation conditions
called conjugation. Cyclins are proteins that regulate the passage of the cell through the cell
cycle. A search for cyclin genes in the TGD yields 26 genes encoding putative cyclins. A
review of the literature revealed that cyclins have yet to be characterized. A protein BLAST
search of a particular cyclin encoded by TTHERM_00196590, referred to as Cyc1, revealed that
the protein does in fact have two cyclin domains. This particular cyclin is related to the cyclin B
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responsible for G2/M phase transition in Paramecium tetraurelia, Dictyostelium discoideum, and
Saccharomyces cerevisiae.
Presumably, cyclin gene expression will fluctuate during the cell cycle corresponding to
the processes the cyclin regulates. Based on the time points at which the cyclin is expressed, the
role of the cyclin can be inferred by looking at the cellular processes that occur at those time
points in conjugation (Miao et al., 2009). RT-PCR of mRNA collected from T. thermophila at
several time points before and during conjugation allows for analysis of expression level at these
time points. The results of this analysis can be compared to the TGED expression profile
available for this particular cyclin, Cyc1.
Methods
The TTHERM_00196590 gene was identified at the Tetrahymena Genome Database
(www.ciliate.org) by searching for proteins with the keyword “cyclin” and choosing
“TTHERM_00196590.” A BLAST search with CYC1 sequence ensured that the cyclin gene
identified contained cyclin domains.
PCR primers flanking an intron were generated for the TTHERM_00196590 gene using
Primer3 (Rozen and Skaletsky, 2000) and ordered from Integrated DNA Technology (Coralville,
IA).
Oligo-dT-primed M-MLV reverse transcription (RT; Ambion, Austin, TX) was
performed on RNA collected from control cells and from cells at various stages of conjugation
using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. 1 mL
of cells (2.1 x 103 cells/mL) was collected at each time point and pelleted at 6,000 rpm. The
supernatant was discarded, and the cells were 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
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template for PCR. 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 product was 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 et al., 2004).
Microarray data during conjugation were collected for the TTHERM_00196590 gene
from the Tetrahymena Gene Expression Database.
Results
Primers were designed to flank the second intron of the TTHERM_00196590 gene (Fig.
1). Based on the sequence provided by the TGD and the primers designed, the genomic product
should be 992 bp in length. The RT-PCR product from the spliced mRNA should be 337 bp in
length.
ATGGCTTGCCTACAAATGAGAAATGGTGAATAATCCATGTTTGGAGACTCAAACAAATTATAAAGCAATCAACTTCGTGCTTTC
GGGCGTGAGATTCAAAATATCCCTGACAGTCGCTTTGTAAAAATTTTTTTCATTTTTCAAATTAATTTCAAATATCCATCTCTC
TTATTTTGATGCTTTTAAGCTTTTTTTTAAGCTGCTTCAACTAAAAGAGTTTGATGAAAGCTAAAGGGAAAAGAAGGATATTGT
TTCAAATGAAGCCATAGCATAGCTATCTCTCTTTAAAATTTTCTAACAATTATTTAATTTATAATAGAATACAAGATCTTAATC
AATAAAAGCAAACATGTAGCCTCACCACGCCTAAAAGCTTAGCATGGAATTAGATACAAATATAAATGAATAAGAGAACGGAAT
GAAAAGAAGTTCTCCTAGAAAATTTTCAATGCATGTGAAGCATTCCTCCGAAGTTCCATAAAACCATTAAAAGAGAATGTCAAG
CTAAATTGACACTGTAAGCAATAATGGAACAGAGCGCTCATATTAAACCAATTAAACTGGCCTTTAAAATAAAAATATTGACAT
GAAAAATATAGAATTTGAATTGAATAGGATTTCTAAGGAAGACTCTGAAAAACCAACTTTAGTTGCTTAATACTCAAAGCAAAT
ATTCGATTATATGCGCTAAAGAGAGGTAAAAAAATACTTTCAAGCTCATTTATTTTATAATGACTCAAAATACCAAAAATGTTT
GCCAACTAAAAATCATTTCCTCAGCAAAGCAATTCATTTTAATTTTGTTAAGCATTCATTTTAAAATAAACATTTTTGTTTTTA
ATTTCCATCAAAGAAGTTGCTATTAAAAATTAAAAGTGATATTTTTGCTTTACGCAATTTGAAATGTAAATTGAAATATCAAAT
AAGCAAGCAAGCAAATAAATAAACAAAATCCTTTTAAATTAATAGAAAAATTTAAAATTAAATTGTAATAAGTATTATCCTAAC
TAATATTTTCTTGCAAAATAATTCAAATAGTTTTATTTTTCGAGAGCCAAGTATTTTTTAGTATAATTATTCGACTACTAATCT
CCTGATTTAATTAAATTTGCAAATTAAAAATTGATCACTTTAAAATTCAATTATTCAAAAATTAAATATCTAATAGAGTTTGTA
TCTAAATGTTTTTTAATCGAATTTAAATTATTAACGATTAAACAAACATCTTTTTTAAAAAAGCGGCATGCTTAATTTATATTT
TCTAATTGAGAAATTTCAAATAAAAGGAAAATTATAAAATATTTTAAAATCACTCAATTTCTAAAATTTATTTTATTCGTAAAA
TAAACTAGATTGCCTTCAAAGTCGGTGAATACATGGAAAAATAAACTTAAATCAATGACAGAATGAGAGCCATTTTGGTTGACT
GGATTGTGGAAATACATAGAAAGTGTAAATTGTTACCTGAAACTTTATTTATTACTGTCAATTTAATTGACCGCTTCTTGGATA
GAGCCACATGCACTAGAGATAATTTATAGCTTGTAGGTGTAACTGCTTTGTTCATAGCTTCTAAATACGAAGAAATATATCCTC
CTAATCTTAATGATTTTGTTGAAGCCACCTAGAAAGCTTACAGAAAAAATGATGTTTTATAAATGGAAGGAAGCATTATTTGTG
CCCTAAATTTCAACTTAACAGTTCCCACATCTCTCCGTTTCCTTGAAAGATACGGACGCGTTGATAAACTTGATAAAAAATCTT
TTGACATGAGCTTATATATTCTTTAACTCTGCTTAGTCGAATATAAATTTGTTAAATACTCAGAATCATTAAAAGCTTGCGCTG
CTATCTATCTCACAAATAAGCTATTTAAAAAGAATATTTGCTGGAGTGATGTCCTCACTGGTCACACTTAACATACCGAGCAAT
AAATTAGACCTTGTGCTCTCGAAATGTGCCTTTTACTTCAATCAGCATCAACTAACTAAACCCAAGCCGTTCGCAGAAAATTCC
TTAGTTCAGAGTATAGCGAAGTAGCAACCATACAAATTGAAAAAATGAATCCTTCATAATAAAACACACAATAGCATTCATATT
AAAATAATTAAAATTACCCTCCCAACATTCCTAAATACAACTACAGAGAATAATATTGA
Figure 1. Sequence of CYCX. Introns are shown in blue font. Primers used in RT-PCR are highlighted in
purple. Sequence was obtained from TGD.
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The RT-PCR gel results confirm that the gDNA amplified with the specified primers was
around 900 bp (Fig. 2). The cDNA was around 350 bp in length. From the gel itself, the
TTHERM_00196590 gene appears to fluctuate in expression during the conjugation process with
peaks in expression at hours 1, 7, and 13-16 of conjugation. A faint band is visible in vegetative
C18
C17
C16
C15
C14
C13
C12
C11
C10
C9
C8
M
C7
C6
C5
C4
C3
C2
C1
C0
428S
427S
428V
427V
gDNA
M
427 cells as well.
1000bp
750
500
400
300
Figure 2. Gel electrophoresis of cDNA from RT-PCR of mRNA collected at different points in T. thermophila life
cycle.
Arbitrary Units of Intensity
1600
1400
1200
1000
800
600
400
200
C18
C17
C16
C15
C14
C13
C12
C11
C10
C9
C8
C7
C6
C5
C4
C3
C2
C1
C0
428S
427S
428V
427V
0
RNA Collection Time Points
Figure 3. Relative intensity of cDNA bands from gel electrophoresis of RT-PCR products from mRNA
collected at different time points in T. thermophila conjugation. NIH ImageJ was used to determine
intensity of bands by analyzing pixels of the gel image. NIH ImageJ produces graphs of each lane and units
of intensity are given as the area under the band intensity peak.
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The gel (Fig. 2) was analyzed using NIH ImageJ. The program analyzes each pixel of
each lane for brightness. NIH ImageJ produces graphs of each lane and units of intensity are
given as the area under the band intensity peak. The band intensities calculated by Image J are
graphed (Fig. 3) and provide an expression profile for comparison to the TGED acquired
expression profile (Fig. 4). The band intensities calculated by Image J indicate that there are
moderate increases in expression of TTHERM_00196590 at hours 1 and 7 of conjugation.
TTHERM_00196590 expression is particularly high during hours 13 through 16.
The expression profile obtained from TGED indicates that TTHERM_00196590
expression builds up in starved cells, drops at the beginning of conjugation (hour 0), increases
between hours 1 and 4, 6 and 8, and 10 and 14 (Fig. 4). The highest expression occurs from
hours 11 to 14 of conjugation.
Figure 4. Expression profile for CYC1 (TTHERM_00196590). Obtained from Tetrahymena Gene
Expression Database (TGED).
Discussion
The gene expression profile, RT-PCR results, and knowledge of homolog cyclin function
allow for speculation about the putative function of CYC1 encoded by TTHERM_00196590. It
appears that this particular cyclin plays a part in DNA replication and/or spindle fiber formation.
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The gene expression profile suggests that TTHERM_00196590 is expressed throughout
conjugation with stretches of increasing expression in hours 1-4 (meiosis I and II), 6-8 (postzygotic divisions of the MIC), and 10-14 (genome rearrangement). Expression was the highest
between hours 10 and 14. The first two time spans include the replication of DNA in preparation
for micronuclear division by either meiosis or mitosis. As gene expression increases, spindle
formation is also occurring through the multiple meiotic and mitotic events. Macronuclear
genome rearrangement, which includes replication of chromosomes about 45 times, would
involve high levels of DNA replication.
The RT-PCR results show that TTHERM_00196590 expression is highest during hours 1
and 7 and between hours 13 and 16. Hour 1 includes cell pairing and presumably DNA synthesis
in preparation for meiotic events. Spindle fiber formation would also be necessary for meiosis.
At approximately hour 7, the 2nd post-zygotic division of the MIC is completed and new MAC
development begins. The time between hours 13 and 16 includes genome rearrangement, which
as mentioned above, includes massive DNA replication in the new MAC.
The speculated involvement of TTHERM_00196590 is also supported by the known
function of homologs in other species, particularly Saccharomyces cerevisiae. In S. cerevisiae
the homolog is a B-type cyclin specific to the well-studied Cdc28p. According to SGD, this
cyclin may be involved in DNA replication and spindle assembly. This homolog accumulates
during S phase and G2 and is then targeted for ubiquitin-mediated degradation as the cell enters
M phase. If the T. thermophila Cyc1 is involved in S and G2 as the homolog in S. cerevisiae
suggests, the oscillating pattern of expression observed in the gene expression profile and RTPCR results can be logically ascribed to DNA synthesis and/or spindle assembly. It is possible
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that as the cyclin is degraded, the cell moves from DNA synthesis to the first stages of mitosis
which include spindle formation.
It is difficult to make predictions on the putative function of Cyc1 without further
experimentation. Localization via a Cyc1-GFP construct would provide valuable information as
to where the cyclin is localized within the cell at time points of peak expression. It might also be
helpful to perform a knock-out of Cyc1 and observe the progression of conjugation.
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References
Abramoff, M., Magelhaes, P., and Ram, S. 2004. Image Processing with ImageJ. Biophotonics
International, 11(7): 36-42.
Miao, W., Xiong, J., Bowen, J., Wang, W., Liu, Y., Braguinets, O., Grigull, J., Pearlman, R.E.,
Orias, E., Gorovsky, M.A. 2009. Microarray analyses of gene expression during the
Tetrahymena thermophila life cycle. PLoS One. 4: e4429.
Rozen, S. and Skaletsky, H. 2000. Primer3 on the WWW for general users and for biologist
programmers. In: Krawetz, S., Misener, S. (eds) Bioinformatics Methods and Protocols:
Methods in Molecular Biology. Humana Press, Totowa, NJ, p 365-386.
TGD. TTHERM_00196590 gene sequence. <http://www.ciliate.org/cgi-bin/gbrowse_details/ttgenomic/?name=TTHERM_00196590>. [Accessed 11/21/09].
TGED. TTHERM_00196590 expression profile.
<http://tged.ihb.ac.cn/search.aspx?keyword=TTHERM_00196590>. [Accessed
11/21/09].
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