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.

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 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.

[pic]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 cyclin-dependent kinases. This protein would appear to activate itself and perform its function, 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.

Materials and Methods

Cyclin genes were identified at the Tetrahymena Genome Database () 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; ). PCR primers flanking an intron were generated for each gene using Primer3 (Rozen and Skaetsky 2000) and ordered from Integrated DNA Technology (Coralville, IA). 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 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

[pic] [pic]

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.

[pic]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). 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.

References

Abramoff, M.D., Magelhaes, P.J., Ram, S.J. 2004 "Image Processing with ImageJ". Biophotonics International, volume 11, issue 7, pp. 36-42

Adachi, M. & Cavalcanti, A. R. 2009. Tandem Stop Codons in Ciliates That Reassign Stop Codons. Journal of Molecular Evolution. 68(4):424-431.

Bednenko, J., Noto, T., DeSouza, L. V., Siu, K. W., Pearlman, R. E., Mochizuki, K. & Gorovsky, M. A. 2009. Two GW Repeat Proteins Interact With Tetrahymena thermophila argonaute and promote genome rearrangement. Molecular Cell Biology. 29(18):5020-5030.

Fu, C., Xiong, J. & Miao, W. 2009. Genome-Wide Identification and Characterization of Cytochrome P450 Monooxygenase Genes in the Ciliate Tetrahymena thermophila. BMC Genomics. 10:208.

Gutiérrez, J. C., Amaro, F. & Martín-González, A. 2009. From Heavy Metal-Binders to Biosensors: Ciliate Metallothioneins Discussed. Bioessays. 31(7):805-816.

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Markmann-Mulisch, U., Reiss, B. & Mulisch, M. 1999. Cell type-specific gene expression in the cell cycle of the dimorphic ciliate Eufolliculina uhligi. Molecular & General Genetics. 262(2):390-399.

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(2):e4429.

Nowacki, M., Higgins, B. P., Maquilan, G. M., Swart, E. C., Doak, T. G. & Landweber, L. F. 2009. A Functional Role for Transposases in a Large Eukaryotic Genome. Science. 324(5929):935-938.

Olins, D. E., Olins, A. L., Cacheiro, L. H. & Tan, E. M. 1989. Proliferating Cell Nuclear Antigen/Cyclin in the Ciliate Euplotes eurystomus: Localization in the Replication Band and in Micronuclei. Journal of Cell Biology. 109(4 Pt 1):1399-1410.

Rozen, SS., and Skaletsky, H. J. 2000 Primer3 on the WWW for general users and for biologist programmers. Bioinformatics Methods and Protocols: Methods in Molecular Biology. pp 365-386.

Zhang, H., Adl, S. M. & Berger, J. D. 1999. Two Distinct Classes of Mitotic Cyclin Homologues, Cyc1 and Cyc2, Are Involved in Cell Cycle Regulation in the Ciliate Paramecium tatraurelia. Journal of Eukaryotic Microbiology. 46:585-596.

Zhang, H., Huang, X., Tang, L., Zhang, Q. J., Frankel, J. & Berger, J. D. 2002. A Cyclin-Dependent Protein Kinase Homologue Associated With the Basal Body Domains in the Ciliate Tetrahymena thermophila. Biochimica et Biophysica Acta. 1591(1-3):119-128.

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