Leann Murphy
Gene Expression Profiling of TTHERM_00332170
Abstract:
Ciliate sexual reproduction, also known as conjugation, includes a series of mitotic and meiotic events. Conjugation proceeds in numerous steps that include nuclear division, nuclear fusion, DNA destruction, and DNA amplification. Each of these steps proceeds in a very predictable fashion. Cyclins have been identified in model organisms as proteins that enable cells to pass checkpoints throughout cell division. It is probable that cyclins are responsible for similar functions during ciliate conjugation. In this experiment a gene from Tetrahymena thermophila (TTHERM_00332170) was obtained from the Tetrahymena genome database and RT-PCR was run. This gene was renamed CYC8. The TGED gene expression for this gene was collected from the tetrahymena gene expression database. The expression profile was then compared to the RT-PCR results. The probable function of this gene was then assessed by integrating expression pattern and sequence comparison data. Results from RT-PCR and the expression profile from TGED show slightly different patterns. CYC8 expression is greatest between hours 4 and 7 of conjugation. These time points correlate to completion of meiosis I and II, selection of meitotic products, mitosis, nucleus exchange, fertilization, and division.
Introduction:
Ciliate sexual reproduction is a multi-step process that occurs over a length of 18+hours. During sexual reproduction, conjugation, two ciliates pair up and after about 3.5 hours, their micronuclei undergo meiosis. Three of the resulting haploid nuclei disintegrate, and the fourth duplicates via mitosis. One haploid nucleus in each cell then migrates (via the cytoplasmic bridge) to the other cell and fuses with the remaining haploid nucleus. The old macronucleus is destroyed and a new one is formed from the micronucleus. Formation of the new macronucleus occurs in a series of steps. First, the micro nucleus undergoes mitosis. Polytene chromosomes are then formed by repeated rounds of endoreplication. Junk DNA must be removed by RNA interference before the chromosomes can be broken up into many pieces (generally into single genes). These minichromosomes are then replicated numerous times and represent the contents of the macronucleus(TGD). In this way genetic information is transferred amongst organisms.
During each hour of conjugation, cells must produce different proteins to proceed through each step. Knowing when each protein is synthesized can enable one to better understand the function of that protein. Cyclins regulate the cell cycle and are found at different concentrations during different times of the cell cycle (Cole, 2001). Although the tetrahymena genome is sequenced, more information is needed to understand the particular function of each gene. By quantifying gene expression during conjugation and comparing the analysis with conjugation time-tables, the putative role of the cyclin protein can be determined.
In this experiment, a prominent event that occurs during increased TTHERM_00332170 gene expression (CYC8) is haploid mitosis (5.5 hours into conjugation). Investigation into the putative function of this gene is beneficial because there are numerous diseases that arise from mutations of mitotic cyclins. Such diseases include cystic fibrosis, Angelman's syndrome, and Liddle syndrome (Schwartz, 1999).
Materials and Methods:
The Tetrahymena Genome Database was used as the source of the cyclin gene TTHERM_00332170. The Tetrahymena Gene Expression Database was then searched for conjugation microarray data. Microarray data indicated the fluxuation of gene exprssion during conjugation.
Primers we made to flank an intron within the gene. Tetrahymena cells during vegetative growth (control), and at all hours of conjugation were collected. The RNA was then isolated and oligo-dT reverse transcriptase was then performed. PCR product was then run on an agarose gel .
Results:
Gel electrophoresis was performed on EtBr stained samples from RT-PCR. The bands were then qualified in order to determine gene expression levels during different stages of Tetrahymena conjugation for CYC8. Higher levels of transcription translate to more of the protein product being involved. Transcription levels were quantified by the intensity of the bands using ImageJ. Transcription levels at various time points are seen both in Figure 2 and Figure 4. Gene expression appears to be greatest at about the 6th hour of conjugation according to the RT-PCR analysis and about the 5th hour of conjugation according to the gene expression profile on-line.
As a control, genomic DNA was added. Figure 3 shows that the reverse transcriptase was successful, and there was very little/no genomic DNA contamination.
Discussion:
The on-line gene expression profile shows TTHERM_00332170 was most highly expressed during the time period of 5 hours into conjugation (Fig. 2). This time period equates to the completion of Meiosis II and the selection of meiotic products. This gene is specific to this time period as it shows only one spike in concentration throughout the entire process of conjugation. In addition, the background level of expression for this gene is very low. The relatively narrow peak indicates that the gene starts to be expressed at 3 hours; it then peaks between hours 4-5, and then drastically subsides by hour 6. Unfortunately there are a lot of processes going on within those brief three hours, and so it is difficult to say with certainty the functionality of this specific protein.
At hours 4 and 5 post-conjugation, tetrahymena are undergoing meitotic events (Meiosis I and Meiosis II respectively). It is likely that this cyclin is involved in nuclear fusion and meiosis. Current research at Columbia University suggests that mutations of a meiotic cyclin (CycA1) have a direct affect in myeloid leukemia. Cyclin A1 is necessary for male germ cells to progress through meiosis. Researchers created transgenic mice in which cyclin A1 was expressed under the direction of the human cathepsin G promoter in myeloid precursor cells. The transgenic mice eventually developed acute myeloid leukemia. Further investigation of CycA1 will provide insight into the development of AML and will aid in the design of new highly tissue-specific molecules for pharmacological intervention (Wolgemuth, 1997).
In contrast to the meiotic events occurring during the 4th and 5th post-conjugation hours, zygotic division is occurring at hour 7 of conjugation. The results from the RT-PCR indicate that the gene is expressed more frequently throughout the conjugation process with the greatest spike in concentration occurring at hour 7 (Fig. 4). Because gene expression is also high at 6.5 hours during the first post-zygotic division, this gene could also play a role in the division of an early ciliate.
>THERM_00332170(gene)
ATGTTTTCAAGTATAGCATCTTCAGCAAAAGGGAGTTAAAACAAAGAAATAAAAATCCCAGTTGGAAATTAATCATAAAATTGTTACAATTACTAATAGACTTTCCAATCACAATAATAATTATAATAGCCTCAATAAGCTTAACCTTTATAGCAAGGCAATTAACGAAAGCCCTCTATATCTAGTGTTGCGAGACCTTAGAAATTTTCTATTTCTATATAGgtaaaatatatttatgaatattgattatgtaaactattaagtctaaaaccatgcattttttaataaacattcacattaagacttaatctatattattttaagattgaacgtttgagtcaataaaaaatctcttaagtattttaaaagttgaatttgaaaaattgcatgctttttaaccttaataaattacatttaatttttaaaatttacaaaaatgcattaagattgaaattatgttgttaaaataatttttgaagacaagacatacaaaaaattatcaaatataagatagaaaacttaataattattaaagctatttttaaattaattttagaataaagaccagTCTTTGCAATAAGAAATACTAAGTGATGATAGAAATTTAACTTATCATAAAAAAAAGCAATCTTATTAGCAGATGATCAGCTCAAACCATTAAGGAAGCATTTAAAACCAAAAGAAGAGTAAGCAAAGCATTAATTTAAACAATCTATAAAATCTTAATTTAGCAAGTACAAATAACAACATTATAAAGGGCTAATAGAATTAATAGAACTAATAAACTGCTTCGACATTTTCTATTCAAAATGCAAAAAATTAGTATTAATAATCACAATAATAATTTAGCTCTGCAACTCTTAACTCGCAAAACAAAAACTCTGCCAAAGATTTTATTAATTATAAAATCCTATAAATACAGGCTAATAGTAGTAAAAATTAATCTATTTCTAACGTTTAAATGAATGGGAATTTACAGCCTCAAAATTAAATTCAAACCTAAATGAATGTCTAAATTTAAGGTAAAAGCAATTAATTCCTTCAAAATCATGGAAGTGGAAATTCCAGTCAATAGCTTGTAATCACAAGCGCTAGACGTAACGAAAGTTCTTCAGTCAATGTAAGATAAAGAGGAAACGAATAAATAAAAAACTAAAATAACCGCTAAGATTAATCTGACTTGAAAGACTAAACTTTAAATCAATATGAAGATATCTTAAAAGAAAAGTAAAACATTTTGTAGCATCATTAAAATACAATGAATTAAACTCAAAGCAAAATGCAACCCTCATAATAACAAGTTATGATTAACAATTATACTTACCATACCAGTCAAAACAATTTAAAAAATGTAAGCAGTAATTCATAATAATCTTAAAATATAAATAACTATCATTAAGTTAGTAACATTGAAAATAACATCAATAATGCAAATACATAGTAAATTTAAGAAAGACCTAAACAAAATTCTTAATAATAAAATATTGGCTACTAAACCACCCACAATAATCATAAAAGTATTAACATCTAATAATCATAAAATTAAAATATAAATAATTACAATTGCAATATTGAAAATAATATTAATAGTGCTAATTTGTAACCATTACTTGAAAAAAATAAATTTAATGTTTAATAATAACACTTTAACAGTAATACTCTGAACTCATATTCCTTTCTCAGCAACAACAATAATATTAGCAGCACATCTAATTTTTCTTCTTTTGGAACACTTCCAAAAGGCACATCAGCTAGTATTTTGAAATAATAATAATAATAATAACAATAACAATAAAACACCCAAACAACTATTAATTCTATGCTATTGCATTTATAGTAAAATGACTTATAAAAAAATTACTTTTTAAATTAATAAAAAATTTAAAATAGCAGCAACAACGGTTAGAATTAATATGCATATTAAAAGAATAATCAATTAGAAATCAATTCTGCTTCTAATTTGGCTCTAACAGATTAAGATGCTTTGAAAGAGTTACTTAGCAAAACTACTTCAACAAAGAGTGCAATCAAAAACGCTGCCAACTACTTCTCTTCTCAACAACATAAATAATAAGTTTAAAATAATATAAATCTTTAATAAGAAGAGAAACCTGGATTTTAATCTGAGCGTCTTAACAACAACAACAATAATACAAACAATACTTAATTTATTCTTTAAAACCAAGTTGCTTAAAAAAAAAATGAATGTATAAACAATTAAAATAAAATCAATGTTCAATCTCACTTAAAGGTTTAAAATTAATAAAAAGCTTAAAACAATCCTAATAATTCTTAAGCAGTAGAACAAATGTACTAAAACTAAGCTCCCATCTAGAGAAAAATACAAGAAAAGATACAAAAATTTACATAAATATAAACATAAATTCAAAACTATAATCCAATCTAAACCTAAAATCTGACATAAAATTCTAATAAATCTCATTTAAAAGCAAACCCTATTTCTATATTGTCTTAGGTAAACTCGTATGGAATTGTAAACAATTCAAAGTAGCCAACACCAGCAATACCCTCAGTTTCTTCTGTTAGCAGTACTTAAAATCTTCATTAATAAAAAACTAATGAAACTCTTAGCAATTTTACTGTTTCTCCAAGTGTTCCAAGTACTTAAGTGCAAATTACAAATCAAATAGGATTATTGAGCTCTAATTCAAATAATAGTACTAATAATAACAATATAAATAATAATTATTATAGTAATCTAAGTAACAAAGCCGAATATCCACATATTTAAAATAATTAAATCGGAGTAAACAATATTAGTTAACTTGGATAGTTGTCTGGATAATGTTCTAGTAATAACAATAACACGACTCAAAATAGTGCCTTAAATTAAAATAATCATAAAAATTTGCTTTCAACTGCTATAAAATCCGAAACTACTCAGGTTTCTAATTTTAGTTAAGATTCTCCTCTTGTATGTAATGAAAATCTATCAGGAATTCTAACAAACAGATATGGCGATAATTCATAATTTAAAAATTTACTATCCTGCAAAAGCAACAACATGTCAAGTAATAATATCAATCCAGGAACTTATAATAGCAACACTTCAAACAATATACACTAAAATTCATCATCTTTTACTTAAACTTTATCAGAAAACAAAGACAAAATACAAAATAATATTTAAATTTAAAATTATATTTCTACTTAACATTAAACCTCTCAAATTAATTCAAAGATTGGAATCACTAATATTTAATTAATTGAAGAATACGATAATTCGATTCCTTATAGTCAATCATAGTAATCTTAAATTTTAAATAATTATGGTGCTTAGCCTAATAATGCTTCTAACAGTTAAAGCAATAATAATAATAATATTAATAATAGCAATTGTGGATTTAAACCTACTCATAAAAAGAATTTAAGCAATCATCTTATAAATAAATTAGGAGCTATTTGCAAAAGTGCCACTGAAGGATCATAAACCTATACCTAAAGTTCTCAAAATAACTCTTAAAACAAAGATTCTTTCAACAATAACAACAACACTTTAAGCAGTAATTAAAATAAAACTAAAAATAATACTGTTAGTATTAGTCTAAATAACAATAACACTAATAATCTTTAAAGTAATACTTTGAGTTAAGTCCAAGATTTAGATCTCAAAAAAGAAGATACTGTTGTCACAAATTATGAGTTAGAAGTTGTGGAAGAAAGCTTAAAAGATAGCATTGAGCAATTTAGTACTAAATAAAATGTTTAAATCAACAAAATACCAAATAACTTAAAATTAGATAAATAAAAAAATAAAGAATAAAAGGAAAGTCTTGAATAGCAGCTTTTAAATTTCTAGCATTAAAATATCTTAAGTGAGAATTTCGAATACAGAATTAAGGATATGAAAGAATAGGAAATAGACTATCTGCCAAATCCAGATTATTTTGATAATTAAACAGAAATTACTTGTATGATGAGATGCATTCTATTTGATTGGATGTTTGATGTTTGCATGAGCCTCATGCTAAAAAGAGAAACTGTTTATCTTTCTTTAAATTATGTTGATAGGTATCTCAGCTAAAAATAGGTTACAAAGCTTAACCTTTAACTTGTTGGAGCTGTTTCTCTTTATATGGCTTGTAAAATAGAAGAAATTCAACCTCCTTCCATTTCAGAGTTTGCAAAATGTACTGATGATGGTTATACAGTAGCATAAATTgtagaatatgaactgctaatgttaaaagtaattaatttattattaaaatatcttttcattcaaataaattttaattagcttatataaaataacaaaaataataaataccttacttacttattaattaattaattaattaataaagcgatttatctccaaaagttttttaaaatacaaatgaatttgtttggaggaagataaaaattaagaataaattagaaattacaataataattaagataaaaagcaattttttaaaaacttttaatttagatagttcaaaaaatatatatgaatatttaaaatatttttatttttaattattattaaataaataaaaataaattataataataaatagGCTTTTGATTGGAAATTAAATCCACCAACATAAATAACTTGGTTGAACATGTATACTGAGATATGGGATAGATTCATAAAATCTAGTTTTGATAAGCCAAATAAATATCAAATAAATTTTGTTTAAGATCAAAAGATTTCAAACAAGAATTACTTAATTCTCTACAGAATTCCTGAATAGAAAAGTTACATGCTTTTTAGATAAATGGTGTAGATTTTGGATTTAATGTAATTAGATGCTAATGTATTAAGATTTGAAAGCAGAATGCTAATTGCTTCATTAATGTACTTGTAGCTAGGCATTTATTATAAATAATTCACTAAAAAATAAGTTCATTCAGGATTCAATGAAAAATACTTATTTGCTAATTCACTTAATAATTTCAACGAATACTTTAATTCTTTTTTGACTCAGCACTTCTAATTTAATCTGATTGACATACTTTCTACTTTGCAACATGTAGCAACTTTCTTTGACGTAGAATATAATTATTCACTTTCTCCAGCAGCCTAAATGATTGATCCTACAAATAACGAGGTAAATATTATAATTCACTTTAAAAATATATAATTAAATAAATTTATTTATTTATTTCAATAGATGTCTTATGAAGAATATTTATCCTATTAAACTTATTTCCCTAATGGTATTGATTATATAAGGAAAAGATACTGCTCATGA
Figure 1: TTHERM_00332170 gene with introns (red) and primers (yellow)
Figure 2: TGED expression profile
M G V V S S 0 1 2 3 4 5 6 7 M 8 9 10 11 12 13 14 15 16 17 18
Figure 3: Gel electrophoresis of cDNA from RT- PCR of mRNA at different time points during Tetrahymena thermophila conjugation. G =genomic DNA; V =vegetative growth; S=starved
Figure 4: Intensity plot of RT-PCR gel electrophoresis (intensity calculated by ImageJ)
of CYC8 with vegetative growth and starved phases. Conjugation of CYC8 is represented by hours 0-18 with time period 4 excluded.
References:
Cole, E., Virtue, M., Stuart, K. (2001). Development in Electrofused Conjugants of Tetrahymena thermophila.The journal of Eukaryotic Microbiology 48(3).
Conjugation: Mating in Tetrahymena thermophila. Retreived Nov 30, 2009. From Tetrahymena Genome Database website:
Schwartz, A., and Ciechanover, A. (1999). The ubiquitin-proteasome pathwayand pathogenesis of human diseases. Annual review of medicine 50(1).
Wolgemuth, D. (1997)Role of cyclin A and mammalian meiosis. Unpublished data