Gene Expression of TTHERM_00433390 During Cell Conjugation in Tetrahymena thermophila

Andrew Ruddy

December 7, 2009

BIO 464 Fall 2009

Bradley University Department of Biology

Abstract

During conjugation of Tetrahymena the cell follows a unique series of meiotic and mitotic nuclear divisions that closely resemble the events that occur in eukaryotic cells. Cyclin genes when expressed are used to drive the cell cycle events in eukaryotes through use of cyclin dependent kinases. Data was collected through use of RT_PCR on the expression of TTHERM_00433390, a possible cyclin gene, found in the genome of Tetrahymena thermophila. TTHERM_00433390 was named Cyc10. Public databases were also used to obtain microarray data on TTHERM_00433390 for comparison. After performing the RT_PCR, it was found that there was little comparison of the data to the obtained microarray. In the microarray data the expression of TTHERM_00433390 reached peaks at hours two and six. The RT_PCR data showed a fairly constant over expressed profile. Because of the great difference it is difficult to create a hypothesis on TTHERM_00433390’s function until further testing is completed.

Introduction

Tetrahymena thermophila is a common freshwater ciliate protozoa that is used as a model organism in biomedical research. Tetrahymena’s most powerful feature is the fact that it has nuclear dimorphism (1). This dimorphism allows Tetrahymena to posses two different forms of its nuclear genome. The micronucleus (MIC) is area in which genetic information that will be passed on through conjugation is stored. The macronucleus (MAC) serves as Tetrahymena’s somatic nucleus in which the MAC is actively transcribed and determines a cells phenotype (2).

In response to stressful conditions, starvation, conjugation will occur in Tetrahymena’s different mating cell types. It has been found that the nuclear events occurring during conjugation in Tetrahymena are similar to the events that occur during meiosis in eukaryotes (3). The events of conjugation also occur fairly quickly, usually in a range of about 14 hours. Figure 1 shows the basic outline of the conjugation cycle of Tetrahymena.

Figure 1. Events in the Tetrahymena thermophila conjugation cycle. Taken from Miao, et al., PLoS ONE. 2009; 4(2): e4429.

At the beginning of conjugation, hours 1-6, the two different mating type cells undergo meiosis and through use of a pilius, the haploid nuclei are exchanged. The exchanged nuclei undergo further division, usually between hours 6-8, and as a result a new MAC and MIC are created. Following conjugation the cell will search for a new mate in order to transfer its genomic DNA. When a new mate is found the MAC of the cell is destroyed allowing conjugation to begin and the MIC undergoes meiosis in preparation for haploid exchange. If Tetrahymena cells are no longer starved, conjugation will come to a halt and the cells will undergo mitosis.

The ability for the cells to undergo mitosis and meiosis is due to the regulation of cyclin proteins. Cyclin proteins bind to a corresponding Cyclin-dependent Kinase (CDK) and controls the activation of hundreds of proteins used in the cell cycle. CDK perform this task by phosphorylating select regions of proteins which in turn activates or inactivates the targeted protein. During the cell cycle the CDKs are grouped into one of three classes: G1 Cyclin-CDKs, S-phase Cyclin-CDKs and Mitotic Cyclin- CDKs. The G1 Cyclin-CDKs serve as a control that moves the cell into the S-phase. These CDKs are used to phosphorylate and thereby activate the many genes used in chromosomal replication. S-phase CDKs become active due to a phosphate being applied from a G1 Cyclin-CDK. Once activated the S-phase CDKs interact with complexes located on DNA’s origin of replication. Once the origin of replication is phosphorylated, replication is triggered which signals the start of S-phase. The last group of CDKs, Mitotic Cyclin-CDKs, creates interactions with hundreds of different proteins to promote chromosomal condensation, formation of the microtubules, and the other essential parts needed for M phase completion. The activation of each type of CDK creates a checkpoint within the cell cycle. In order for the cell to move onto the next stage of the cycle the current CDK and must complete all of its required tasks and be degraded before the next stages CDK is produced.

For this study 15 possible cyclin genes in the organism Tetrahymena thermophila were selected and their expression profiles were obtained. The determined expression profiles were then compared to predicted expression patterns that were obtained from microarray data.

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 (2) 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 (4) 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) were 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 (5).

Results

Bioinformatics

>TTHERM_00433390(gene)

ATGTTTTAAAATACAAATTTCAACTGCAATCAGGAAGGTTAAAGTTAGAGAATTAATTCGAATGTCTAGGGCTCAAGTGGTAAAGAGAGGAGATAACAGAACTAATCACAGAAGATGAACAATTAATAAAAAAATGAGTAGAGAAGCAAGTAAAAAAATAGACGTAGCAGTAGAAAATCTTAGAGTAAGAATTCAAATAATAATGCTACATAGATAGAATAGCAAAACTAGTAATAAGAATTGATAAATAACTAAAGAGTAGAGAGCGCTTAAAATTCCAACAAAAAAATAGGATAATCTACCAATTCTACTTTATCGTCCTCCATTTAATCATCTCCTAGTTCTTATACTTCATAGTTTCCTTCTATAAGTGAAATTACTTAGTTAACTGTCCTTAAAGGAAATTCAAATAATTATACAAATAAAACTAATTTTTTAAGCTCTCCAAATGAAAATTCATACTAAGATTCACCTGCTTAAAATCAATAGCAGCGAAGTGTTTCTCCTGTCAAAGAAGAGAGAAAGTATGATTCAGATGATCAAGAGAATAACCCAAGTCTTGCAAACTTATAAACAAGCAATACAAATAACTCCATAAACAATATCCAAAATAATAACAACATGAATATTCATATTAACTAAATGAATTAAAGTATCGCAAGTAATAATAACTTAGGAATTTCAAATTTCATGAATGGCAACAGTAGCCAAAGCAATCATTTAAGCCACATTCACTAGCACCACTCACATAACTTACAGCAAATGTGCTGTCCTTTAAATAACAGTAGCAATTTGAACAGTATGATAAGCAGCAATAACTTTGATATTAATAACACTAGTAGTTGTCATGAGAAATCTAGTTCAAAGCAGAGAAATGTAAATGGAAAAGGCAAAAAATATGGATATAAACATGGATAAATGAATCTGAACTGTGATTAAAATAGTTTGAATGGTAAAGAGCAGCACAAAACATGCCAAATAAATTGCGAATAAGCACTATGCTAGAATAATAAAAGCTTAAGAAATTTATCAAACTTACTAAAAAGCTTGAAATTTTCACCTCTACTTGCTTAATAATAGTAAGTAACATAGAGCTTAATGACTTTACCCTTCGAACTTCACTTCATTATTTGTTCTTATTTATCTCCCAAAGATATTTACCTGCGCCTTGCAAATGTAAATTAGTACTTCCGTAAAATGTGTAAAGATACTCGTCTTTGGAGCTATATCTAGAAGTTTTAGACTTTGACCATTAATGACAAATATTAGAAGAATATCCCTGTAGTAGAACGCAGATCCAAAGGAAGACTCTTTTCAGCAGTATCCCGTCTAGATCATAATCAGgtaaaaaataaattgtccttcattttaaattttctatttcaaattttaaatcaatcaatcaatcaatcaacaaacatatttgaaaacaaattcttggttgaaaaaaccgaaacagattaattcttttaaatttttgaacgaaattcatttttttaatgttcaattaaattaaaaaaaagTTTATGATTAGAAACGTGAACATTATGATAAGCAATGCTGGATAAGATGATGGAGTTCCTACTAGTATTTTAAGAGAACTCAGCTATTTGTAGGCTCTTGATCACCCAAATATCACAAAgtaattttaattattaattcattaacaagtgaaaaactactaaatatttattcattattgtattctgctcttttcaaatcagaaaaaattatcttaaaatcatggagattttcataaataaactgaaaaaatattctaaaatattaaatttttatttaatataaaaatagAATTCACGAAGTAGACATAAAAAATGTGATAGTTCAAATAGTTACAAAAAGATAAGAGTATAACTTGAAGGAATATACAAGAAGATATATCAGCTAAGTTCAGGATGACAGTAAGGGACTCACTCGTCCTCAATACAAAATTCCTTTGATTAAAGTTAAGGAAATTGCTTACTAGATTCTTGAAGGTTTAAATTACTTGCATCACTAAGGAATTATGCATAGAAATTTAAAGATTGATAATATTTTGCTTGATAAAGATGTTGTAAAAATATCAGATTTCGGTCTTTCAAGACTTGTTTCGATTCCCCACATACCATACACTCCTGAAGATCCTAAAGAAAGAGAACGCTCAGGAAGAGAAGCTCGCCGTCTTTGGTACAGAGCTCCTGAGCTCCTTTTGAGAAAAAGCATCTACACCTTTGAAATTGATATGTGGTCTTTTGGCTGTCTTTTAGCTGAAATTGTTTTGAATGAACCTTTATTTGCTGGAGgtaaaaataaataaaataaaaattaatttaaaacaaatagaattttatacaaaattttttttgagcatttacaactaattaattacaattgtttttatgaaactgatagcaatcaatatattaaatgtctttaaaagcagataaatttacaaataatctagatttaggatagctttctaaattaatatcaagcataagttaaaaacagaaaatttaaatttgaatgcaaattcataaaattaagatattaaggcaacaaacaattatcaatttaatttattttattttttgttgttttttttcactcaaaaagaaaaatagataaaatagacaaaataaatgattgattttcatcatatataaaaataatacttgttaaattgaattagatccattcaaatgacaaataaattgattaattctttttatactaaatagATTCTGAAATTGAGTAGCTTTTCAAGGTATTCAAATTTATTGGTTCTCCAGATTCAGAAACCTTAATGTCAATGTGTGACAACCCTGATTACTAGCAAACCTTCCCAAAATGGAAAATGATCAATTTATCCCATATCTGTGAATAAAATTAGAGTGAAGAATTCAAATAATTAAAGACCACAATGATTCCCAACAGATAGAACGGATTTTAAAAACTTAAAAATTTAGGCACTATCTTGGGAATGTAAGGAATGTAGCTTTTGAACTCATTACTTCAATTAAATCCTTATAAGAGAATATCTTCTCAAGAGGCTCTTAACCATCCATTCTTCGATGAAATAAGATAGTAAAGAACTTTAATGAATATTCCCATGCAAATTGATGAAACCATTAATATTAATAAGTGTTGCAGCTAAGTAGTACCTTCATGCTATTTTTAGTTCTCACTACCTTACTGCCATTTATCAACAATTTATTAAACTATGGTTAAATAAGAAAAGATAGAAACCTTGATCAAACCTGATTACATGAAAGACTAAGCCTTAATCACTGAAAATATGCGTATAATACTTATTGATTGGCTAACTGATGTAAGCGTTCATTTCGAAGCATAAGACGAAACTCTTCATTATTGTATTTCTTACATCGACAGgtaaataaaaaccaaatcttcttattttttttaagttataattttattttttaaaataattattttaattaaacgtctttaattaatctttactaaattaatgaataatattataaatcaaaagAGCTTTGGGTATCATGAATATTGATAAACAAAAACTGCAGTTAGTAGGAGTAAGTTGCATGAAAATTGCAGAgtaagaaattaatttaataataaagaatttattaatatttaaaatttaaaaatagTGTATTTAATGAGAGATCTAGAGAATATTACAAATAAGAAAACGCTATCGAATATGCATACATAACAGCAGAAGAGTATAATGCAGCTTAACTCATTGCTATGGAGAAAAAAATTCTAAAAGTTCTCTCTTTCTAATTAAATACTCCAAATATGATGTATTTTTTAAAAATAATGTGCACTTTATTTGATACTGACTCCTAGGTTTCCGTTATTGCTATGgtaaattatttattaataaaataaataaatcgtataaaaaataaaagcaaaaattatactgattactttttattatgttttgcttttatttttttcaatattattttagtttaaaattttttgatttttaatttaaatttatatttgaaaattaaaagTTCCTGGCCGACTTGCTACTTATGAGTTACGAAGCATTGAGATTTAAACCAAGCTTACTAGCCAGTTGCTGTATATTTTTGAGCTTTTTGACTAACGAGAGAGAACTTCCTACTTAAGAAAAGCTAAATGAAGTTCGTAGCCTTTTAGACCATTATACTATTAATGAGTTTAAAGAAGCAGCTGAATTTGTTAGAAAATTCTGGCATTATTATAGATCTGATCCCAACACACTAAACTTCTAATCAGTCTACAATAAGTATGCTGTTGTATATGGTCTAGAAGCAAGACTAATTAATGCTCCTATAATAGAATAAGCCTAATACAGCTAATGGATCTATACAAAATGA

RT-PCR

RT-PCR Intensity Graph

There is no data point for C4. The reason for this is that there was a error in the collection and treatment for the Tetrahymena at the given hour.

Microarray Expression data

The microarray expression profile was obtained from the Tetrahymena Gene Expression Database.

Discussion

The RT-PCR data of Cyc10 (TTHERM_00433390) does not match the expected intensity that was obtained from the Microarray Expression. The one area where the data is relatively close is at the hour six and seven marks. Between this time period in both the RT-PCR and Microarray data, intensity reaches a maximum. At all other time periods however the intensity gained by RT-PCR is much higher then what was found in the Microarray. Due to these great differences it is very difficult to try and make a hypothesis on for the putative function of each of cyclin TTHERM_00433390. If you look at just the microarray data the cyclin seems to be involved somewhere in the G or S phase of mitosis. The reasoning for this assumption is that at hour 2 the cells have paired together and are beginning to undergo division, and a little after hour 6 the cell is beginning to undergo its first postzygotic division. The RT-PCR data however, shows the cyclin to be almost constant throughout conjugation. The RT-PCR experiment should be repeated at least one time to see if better results can be obtained. New primers may have to be created for the experiment as well.

References

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MA. Microarray analyses of gene expression during the Tetrahymena thermophila life cycle. PLoS ONE. 2009;4:e4429

3.  Cole ES, Cassidy-Hanley D, Hemish J, Tuan J, Bruns PJ. A mutational analysis of conjugation in

Tetrahymena thermophila. 1. Phenotypes affecting early development: meiosis to

nuclear selection. Dev Biol. 1997;189:215–232.

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