Molecular Biology – Final Laboratory Report



Catherine Marek

Cell Molecular Biology

12/8/09

Gene Expression Profiling of TTHERM_00293270 in Tetrahymena thermophila

Abstract:

A BLAST search for cyclin in Tetrahymena thermophila identified TTHERM_00293270 as a cyclin. A cyclin contains a cyclin domain and regulates the cell cycle of Tetrahymena. By using the Tetrahymena Genome Database (TGED), an expression profile was obtained for TTHERM_00293270. We first hypothesize that TTHERM_00293270 is a cyclin gene and named it CYC7. In addition, we hypothesize that the gene is expressed at varying timepoints to control the cell cycle.

Introduction:

Tetrahymena thermophila is a ciliate commonly used as a model organism in molecular biology. T. thermophila are unique due to their two distinct nuclei, the macronucleus and micronucleus. The macronucleus (MAC) is responsible for somatic processes, while the micronucleus (MIC) contains the DNA for conjugation (Eisen et al, 2006). In the ciliate life cycle, conjugation is the stage for sexual reproduction. During conjugation, T. thermophila undergo division to divide its replicated genetic material (Martindale, 1982). First, cells and chromosomes pair up. The micronuclei proceed into Meiosis I and II. Four haploid pronuclei are the products. The old micronucleus, as well as three pronuclei begin to disintegrate. Only one pronucleus is not destroyed. As the sole survivor, this pronucleus divides into two gametic nuclei. The division creates migratory and stationary pronuclei. Next, pronuclei are exchanged and the zygote nucleus undergoes fertilization. Finally the zygote nucleus undergoes two postzygotic divisions. A new macronucleus develops.

To proceed through life cycle stages, such as conjugation, ciliates utilize cyclin genes. The expression of cyclins is regulated through varying concentration levels to signal for the end of one stage and the beginning of a new one (Galderisi et al, 2003). Named accordingly, G1, S, and mitotic cyclins are essential for progressing through the cell cycle. Cyclin dependent kinases are the enzymes that are activated by cyclin genes. G1 cyclins prepare DNA for replication by activating certain transcription factors. These transcription factors activate the genes necessary to produce DNA polymerase, nucleotides, and S cyclins. DNA replication occurs when S phase cyclins activate DNA polymerase. Mitotic cyclins are responsible for chromosome condensation, nuclear envelope degradation, and spindle formation. The ciliate, Tetrahymena thermophila, depends on cyclins for its progression through the cell cycle. By identifying the varying expression of TTHERM_00293270, it is possible to infer possible functionality of such cyclin.

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; tged.ihb.). PCR primers flanking an intron were generated for each gene using Primer3 (frodo.wi.mit.edu/primer3) 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 the manufacturer’s protocol. 1 mL of cells (2.1 x 103 cells per mL) was collected at each timepoint, pelleted at 6000 rpm, supernatant discarded, and cells resuspended in 1 mL of Trizol. 180 ng of each template RNA was used per each reverse transcription reaction. cDNA was diluted 1-5 and used as a template for PCR. PCR was performed using GOTaq (Fisher, Hamptom, NH). 15 μL of completed PCR reaction products were separated on a 2% agarose gel. DNA bands were visualized using ethidium bromide and photographed with a Kodak EDAS 290 imaging system. Band intensities were determined using ImageJ (Abramoff, 2004).

Results

> TTHERM_00293270 (gene)

ATGAATTCCTAAGTAGAAATATAATCTATGCCTTCTGAATAGACCTTCTTATCTTATGAAAATGCCTGCCACGCTGGAGTTTAGTAGAAGTTCTACAACAATTCTTTAGCCTAAAAGAGTGCTGCTGGTTTCCGTGGAGGTGAGTGCATCCGTAAGAAAGCTAAGAACTGTTAAATTAATATTGTCAATCCTCAAAAAGAAATATCTGCTGCTGATTGTATGGAAATTGAAGATGATTGCAATTTGAAGTAAAAATAAAACTTTGAGCAGTAATAGTAGCAAGATTAAAGAACAACCGAGTGCAATTCTTTTTCTGACTACTTTACCAGCAAAAGATTTTAATAAATTTAAGTTCTATTAAAGAGCGCTTCCAACGAAGAAGCTTACTCATCTAGAATAATTTCTTCTATGATTAATgtaaacaattaatttcatttatctacttaataattccatttttaagtatttccgatgacttattttctattgttaatttattaatttacttattaacaaacatatcagcattcaattaattctccccaatttttgaaattcacattcatttgcaaattaaatttttaatcttgatatgcttgcttgtctcacccattattaaatagatactaaattttttaattaattaattaatattttctttggatatgaaaatcaatacaatacaacacaacacaacactatcatcttttaatagcagtattctatctattttgctaattatcatactctttttaagacatatggattacttatttatgatataaaaaatttgaaaaaatctataaatctgattattggattaatccaaatcattatctaaaaatcttaagatttatattcatttttttttataacattcatagAGTTAAAAATCACCAAGTGACTTCTTGCAAAACCACACAATAACTCCTGAAATAAGAGCAAAGATGATTGACTGGATGATTGAGGTTACAACAAGCTATGGTTAGAGTGATTAAACCTTCTTCTTGGCAGTACAATACATGGACGCATTTTTCTAAAACTCTTAAAGgtaattaaaattaatttaaaaaaatattttttgatatcaaagttgattactgatttaaagttaatttgattaacactataacttttcccatattaaattctaatatttttaaaatccgtcacatttctaattctaaatgattaaattagatcatttaatattatttccctccataaagtttgcataaaaacgctaatttattaaaaattaacacaataaagaaatttaaaatttttcaattctttttgataataattattattattttgcattcaatctttccttccttcccgaccttatttaattaattaattaaataaataaataaaaaataattaataatactgataattcctaaataaaaatttttcctttctaggtttttaacatgttttaattttaatattttaaatattaatagATAGTTAACTCCTCAAGATTTACATTTGACTGGTGTTGCTAGCATGTTTATTGCCTGCAAATATGAAGAAATATATCCTCTTCGTCTCTAAACCTTCTATGAAAAAATTGGTCACAAGAAATTGTCTATTTCAGATATCAAGTAAAGAGAACAAGAAATATTGGCCTCTATAGATTTTGATTTAAGTTCTCCCACTATCCTTGACTTTATGGCTGTAACAGTCCAAAAATTAAATTTAGAAGAACACCTTCTTGACACACATCTTACTTTTTTCCGTAAAATGTGTATTTACCTTGCAAAAATGGTATCACATGAATATTCTATCATTAATAACCATAACCAAACTACTATTGCTGGAGCAATTATCTATGTTTCTTTTAAAATCCTTTAATAAATGTACAATGAATTTAATCTTGTTGCCCTTATACAACAAGTTATCTAAATTCTAAGCTTAAACAATGAAGAAATTCTTAAGGCTTCTACTCTTTTGCTTAATCTTGCAAGAAGTTTTGAGAGATAATATCCTAACTTGAATAATTTAAGAAAATTTAGTGCAGATATCCAATAAATAAGTGCTTCTCCCTCTTCATCTTCACTAAAACCAACAAACCAATAACAAAGAAGAGCAAATAACTGA

Figure 1: Gene sequence for TTHERM_00293270, with introns and primers identified. Introns are labeled orange, while primers are hilited in pink.

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Figure 2: Gene expression profile for TTHERM_00293270. The lowest point of expression is at the beginning of conjugation, from C-0 to C-2. Expression is highest during hours 6 and 7 of conjugation.

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Figure 3: Gel electrophoresis of cDNA from RT-PCR of mRNA at different time points of Tetrahymena life cycle. TTHERM_00293270 is transcribed at low levels at the beginning of conjugation and at higher levels 6 and 7 hours after conjugation. Band size is 208 bp from cDNA.

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Figure 4: All timepoints of the Tetrahymena life cycle represented in cohesive graph. Expression of TTHERM_00293270 is highest at hour 7 during conjugation and lowest at hour 4.

Gene sequence of TTHERM_00293270 was obtained from Tetrahymena Genome Database Wiki. After introns were identified, forward and reverse primers were created (Figure 1). TTHERM_00293270’s expression profile from TGED was used as basis for comparison for our own data (Figure 2). Performing gel electrophoresis of the cDNA from RT-PCR results created varying intensities for each timepoint of the Tetrahymena lifecycle. These intensities represent how much gene expression occurs at varying times (Figure 3). Using imagej, the intensities were quantified and assigned arbitrary units of expression (Figure 4A). A new expression profile was created using the data obtained (Figure 4B).

Discussion

Possible functions of the gene, TTHERM_00293270, can be deduced from analysis of its gel and gene expression profile. The intensities of the gel bands correspond to the gene expression profile, however, are expressed an hour later than in the expression profile. Attributing the time point differences to experimental error, TTHERM_00293270’s expression is described below in accordance with its expression profile.

TTHERM_00293270 is initially expressed during the growth phase (S-0 through S-24). As the growth phase progresses, the gene has a fairly linear pattern of increased expression. At C-0, the start of conjugation, TTHERM_00293270 abruptly ends its expression. From analysis of this expression pattern, TTHERM_00293270 may function as a G1 cyclin to promote the transition from G1 to S phase. During conjugation, the gene has varied expression, an indication that it may promote transcription of other cyclins. The gene has a maximum value at C-6, the fertilization timepoint. Post-fertilization, TTHERM_00293270’s expression decreases dramatically. Its decreased expression may signal for proteolysis during the first and second post-zygotic divisions at 6.5 and 7 hours, respectively. Proteolysis has two distinct ubiquitin-conjugation pathways (King et al, 1996). One pathway signals for DNA replication, while the other promotes the transition from metaphase to anaphase. The first proteolysis pathway was likely activated at the end of G1. The second pathway is observed in the two post-zygotic divisions in T. thermophila. These divisions mirror processes that occur in anaphase. Spindle development is key to both anaphase and post-zygotic division. Shortening of the kinetochore microtubules facilitates translocation to spindle poles. Then, extending the microtubule network furthers separation of spindle poles. It is likely that the gene contributes to spindle development. The expression of TTHERM_00293270 corresponds to patterns of G1 cyclin activity as well as proteolysis events at the end of growth phase 1 and post-zygotic divisions during conjugation.

A BLAST search revealed that TTHERM_00293270 has similar sequences to Paramecium tetraurelia. The similar proteins encoded in P. tetraurelia genes function as cyclins that promote cell division and chromosome pairing. Similar sequences found in the Saccharomyces Genome Database (SGD) correspond to the predicted function as TTHERM_00293270.

References

Abramoff, M.D., Magelhaes, P.J., Ram, S.J. "Image Processing with ImageJ".

Biophotonics International, volume 11, issue 7, pp. 36-42, 2004.

Eisen JA, Coyne RS, Wu M, Wu D, Thiagarajan M, et al. 2006 Macronuclear Genome Sequence of the Ciliate Tetrahymena thermophila, a Model Eukaryote. PLoS Biol 4(9): e286.

Galderisi U, Jori FP, Giordano A. 2003. "Cell cycle regulation and neural differentiation". Oncogene 22 (33): 5208–19.

King RW, Deshaies RJ, Peters JM, Kirschner, MW. 1996. How Proteolysis Drives the Cell Cycle. Science, 274.

Martindale DW, Allis CD, Bruns PJ. 1982. Conjugation in Tetrahymena thermophila. A temporal analysis of cytological stages. Exp Cell Res 140: 227–236.

Miao W, Xiong J, Bowen J, Wang W, Liu Y, et al. 2009 Microarray Analyses of Gene Expression during the Tetrahymena thermophila Life Cycle. PLoS ONE 4(2): e4429. doi:10.1371/journal.pone.0004429

Primer3. Whitehead_Institute and Howard Hughes Medical Institute by Steve Rozen and Helen Skaletsky. . (Accessed 10/11/09)

Tetrahymena Genome Database. 2005-2006 The Board of Trustees, Leland Stanford Junior University. . (Accessed 9/10/09)

TTHERM_00293270 Gene Expression Profile. Tetrahymena Gene Expression Database.

. (Accessed 10/20/09).

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