Download Molecular Biology – Final Laboratory Report

Seth Adams
Cellular & Molecular Biology, Bradley University
8/12/09
Cyclin gene family expression during conjugation of the ciliate Tetrahymena thermophila
Abstract:
Conjugation, the ciliate sexual cycle, follows a unique series of meiotic and
mitotic nuclear divisions, nuclear fusions, DNA elimination and amplification, and
nuclear destruction. Expression of cyclin genes is known to both drive and respond to
cell cycle events similar to these in other model systems. In order to determine if cyclins
are involved in regulating the distinct steps of conjugation, we have collected expression
data on the cyclin genes found in the genome of Tetrahymena thermophila from public
databases and performed RT-PCR analyses to complement these data. The majority of
the 23 cyclin genes we have identified appear to be active at very specific points during
conjugation. By integrating expression pattern and sequence comparison data from this
study with studies detailing the cellular events that occur during the 18 hour conjugation
cycle, we are able to make hypotheses about the putative function of each of these
cyclins. This paper focuses on the gene TTHERM_00079530, which we have now
assigned the name CYC2.
Introduction:
The ciliate Tetrahymena thermophila has a different genetic code than the
“universal” code that humans use; in it and other ciliate species (Paramecium tetraurelia,
for example), the codons TAA and TAG are reassigned to produce Glutamine, rather than
function as stop codons (Adachi and Cavalcanti 2009). Adachi and Cavalcanti (2009)
propose that perhaps this leads to “leaky termination machinery”, and they support it with
their findings of a high rate of tandem stop codons in both Tetrahymena thermophila and
Paramecium tetraurelia. They show that the number of tandem stop codons is greater
than expected by chance and they hypothesize that the tandem stop codons are a way for
the cell to minimize effects of stop codon read-through. This implies that proteins in
these ciliates may commonly express extra domains, including cyclin proteins, the topic
of this study.
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Sexual reproduction in Tetrahymena thermophila is a process called conjugation,
which all ciliates use. The cell contains two nuclei: a micronucleus used for conjugation
and a macronucleus used for transcription (Malone et al. 2008). The process involves the
creation and deletion of nearly whole genomes, and is promoted by a suite of proteins,
including cyclins (Bednenko et al. 2009). Many of these proteins are specific to a single
nucleus, as the events in each are very different, therefore the nuclei contain different
transporters to move different proteins inside them (Malone et al. 2008). The two
genomes are combined in a way reminiscent of other sexual reproduction by the
micronuclei, and the macronucleus is replaced with a version comparable to the new
micronucleus (Bednenko et al. 2009). The original genome is not conserved in this
process, i.e. the “parent” cells are no longer present. Their genomes have been replaced
by those of the “offspring”, and a division after the replacement finishes the process.
Miao et al. (2009) provide a diagram of conjugation with time points (Figure 1).
These same time points are used in our research of these cyclins. Miao et al. (2009) also
provide valuable information on this process: two Tetrahymena cells will pair with one
another, forming a link between cytosols. Meiosis will occur in the micronuclei
(abbreviated MIC), followed by deletion of three of the four products. Mitosis will then
occur, creating two identical haploid micronuclei. One micronuclei from each cell is
exchanged, at which point the two haploid micronuclei fuse to form a diploid
micronucleus in fertilization. The new micronucleus will then undergo mitosis twice,
producing four total micronuclei in each cell. Two of these will develop into new
macronuclei (abbreviated MAC), and the cells disconnect. Next, the old macronucleus is
destroyed, along with one of the two remaining micronuclei. When given more food with
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which to power cellular functions, the micronucleus undergoes mitosis once again,
leaving two micronuclei and two macronuclei. This is followed by the cells dividing,
producing four individual cells, each with one micronucleus, one macronucleus, and the
same new genome.
Ultimately, it is still unknown why this process seems so convoluted. Some
deletions and replications seem entirely unnecessary. For example, the micronucleus
deletion between hours 10-14 is followed by replication of the other, matching
micronucleus. The reason behind this and other such events are unknown at this point in
time. If this study allows us to gain insight into the function of cyclins present at that
time, we may be able to determine why such events occur.
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Figure 1. Stages of Conjugation in Tetrahymena
Many things are still unknown about ciliate genomes. A specific example is the
role of transposons. At least one ciliate, Oxytricha trifallax, has a very high number of
transposons, and it appears that they have a function in development (Nowacki 2009).
Ciliates represent a convenient way to learn more about genomics due to their unique
binucleate nature.
Another strange example was discovered by Markmann-Mulisch et al. (1999) in
Eufolliculina uhligi. It has domains that are associated with both cyclins and cyclindependent kinases. This protein would appear to activate itself and perform its function,
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as the cyclin portion binds and activates the cyclin-dependent kinase portion. Our study
has found an analogue of this protein in Tetrahymena thermophila as well, indicating that
whatever event led to this (transcription error, duplication eror, et cetera) happened early
in ciliate evolution.
As one can imagine, the control of conjugation must be very specific, and many
proteins are involved, as previously stated. One variety of proteins that are active, and
are the topic of this research, are cyclins. Cyclins are proteins that promote a certain
phase of the cell cycle, hence the name. They usually work through proteins called
cyclin-dependent kinases (Zhang et al. 2002). They function as all kinases,
phosphylating specific substrates, and they are activated by specific cyclins. The method
by which they act differs, as does the location in which they perform their function (Olins
et al. 1989).
There are many different cyclins in many different organisms. One way to help
explain the diversity among them is through duplication events. Duplication events lead
to entire gene families (Fu et al. 2009, Gutiérrez et al. 2009). In an attempt to further our
understanding of the Tetrahymena thermophila cyclins, a potential gene family map was
constructed.
For many cyclins, their function is unknown. We know that they are cyclins due
to the cyclin box: a conserved gene sequence found in all cyclins (Zhang et al. 1999).
The goal of this paper is to propose functions for cyclins found in Tetrahymena
thermophila. Zhang et al. (1999) used similar methods, identifying when the cyclins
were present and positing functions for them. We create expression profiles for the
cylins during conjugation, and propose functions for each of them based on our findings.
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Materials and Methods
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. 2009) 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 (Rozen and
Skaetsky 2000) and ordered from Integrated DNA Technology (Coralville, IA). OligodT-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
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 et al. 2004)
Results
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QuickTime™ and a
decompressor
are needed to see this picture.
Figure 2. Expression data for CYC2 in T. thermophila. Top, microarray expression profiles for each
gene from TGED. L = vegetative log phase growth; S = hours under starved conditions; C =
conjugation (hours post-mixing). Bottom, RT-PCR analyses. Lane 1 = DNA MW Marker. Lane 2 =
CU428 genomic DNA template (contains intron). Lanes 3,4 = CU427 and CU428 vegetative. Lanes
5,6 = CU427 and CU428 starved 24 hours. Lanes 7-25 = conjugating (0-18 hours post-mixing). RNA
concentrations were standardized using Nanodrop prior to RT-PCR.
This is one set of data from the experiment as a whole, which produced profiles
for 23 cyclin genes. The data gathered from the gel was constructed into an expression
profile, similar to that of the TGED.
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3500
Arbitrary Units of Intensity
3000
2500
2000
1500
1000
500
0
427V428V 427S 428S C0
C1
C2
C3
C4
C5
C6
C7
C8
C9 C10 C11 C12 C13 C14 C15 C16 C17 C18
RNA Collection Time Points
Figure 3. Gene expression profile for CYC2, created based on ImageJ data regarding the gel.
The profile created is clearly similar to that found on the TGED, with the
exception of time point C4. None of the gels showed expression at this time point. We
believe that, in preparing this sample, there was human error that caused this data.
Discussion
Cyc2p appears to signal Meiosis I. It comes on and the chromosomes pair in the
crescent phase. When it begins to drop off, Meiosis I finishes and Meiosis II begins. The
gene also has a small resurgence during exconjugation, the 10-14 hour period.
During that period, one micronucleus is destroyed. There is destruction of
micronuclei earlier in conjugation, however it happens after the protein is already
leaving. It occurs when concentration has dropped by ~50%. It would be a stretch, but
one could say that the “leaving” signal does not occur until that point, and that when the
protein leaves, it signals for micronuclei destruction.
Another possible function is related to micronuclear duplication. This would
assume that the duplication proceeds during the exconjugation period (unlikely at best).
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Even if that were the case, there are mitotic micronuclear divisions around hour 6 in
which the protein is nearly absent.
So, the gene’s function likely deals with something that only occurs in Meiosis,
and not in Mitosis. The processes are similar, but there is a glaring difference that may
shed some light on function: crossing over. Crossing over also separates Meiosis I from
Meiosis II, which appears to start upon the exit of the protein. Crossing over would
involve breaking bonds on DNA strands, which would be useful in destroying the
macronucleus and micronucleus during exconjugation.
I therefore propose that the cyclin-dependent kinase activated by Cyc2p activates
proteins which phosphorylate nucleotides in the middle of DNA strands (actually
phosphorylating the sugar-phosphate backbone), acting as a phosphodiesterase, and
causing the strands of DNA to separate at that point.
Many of the genes analyzed by RT-PCR match the pattern predicted by the
microarray data, though additional expression peaks were observed for a small number of
the genes. Surprisingly, a few of the RT-PCR experiments failed even though the
primers amplified well from genomic DNA and the TGED profile suggested they were
expressed at a high enough level to be detected during our study. In these cases the most
likely explanation is that the primers were designed including a cryptic intron sequence
not identified by the genome sequencing center. New primer sets will be ordered to test
expression of these genes, as well as the seven genes not analyzed to date. Data and from
this project will be submitted to the Ciliate Genomics Consortium website at the
Claremont colleges and will be used to update annotations at the Tetrahymena Genome
Database.
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