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)
ATGTTTTCAAGTATAGCATCTTCAGCAAAAGGGAGTTAAAACAAAGAAATAAAAATCCCAGTTGGAAATTAATCATAAAATTGTTACAA
TTACTAATAGACTTTCCAATCACAATAATAATTATAATAGCCTCAATAAGCTTAACCTTTATAGCAAGGCAATTAACGAAAGCCCTCTA
TATCTAGTGTTGCGAGACCTTAGAAATTTTCTATTTCTATATAGgtaaaatatatttatgaatattgattatgtaaactattaagtcta
aaaccatgcattttttaataaacattcacattaagacttaatctatattattttaagattgaacgtttgagtcaataaaaaatctctta
agtattttaaaagttgaatttgaaaaattgcatgctttttaaccttaataaattacatttaatttttaaaatttacaaaaatgcattaa
gattgaaattatgttgttaaaataatttttgaagacaagacatacaaaaaattatcaaatataagatagaaaacttaataattattaaa
gctatttttaaattaattttagaataaagaccagTCTTTGCAATAAGAAATACTAAGTGATGATAGAAATTTAACTTATCATAAAAAAA
AGCAATCTTATTAGCAGATGATCAGCTCAAACCATTAAGGAAGCATTTAAAACCAAAAGAAGAGTAAGCAAAGCATTAATTTAAACAAT
CTATAAAATCTTAATTTAGCAAGTACAAATAACAACATTATAAAGGGCTAATAGAATTAATAGAACTAATAAACTGCTTCGACATTTTC
TATTCAAAATGCAAAAAATTAGTATTAATAATCACAATAATAATTTAGCTCTGCAACTCTTAACTCGCAAAACAAAAACTCTGCCAAAG
ATTTTATTAATTATAAAATCCTATAAATACAGGCTAATAGTAGTAAAAATTAATCTATTTCTAACGTTTAAATGAATGGGAATTTACAG
CCTCAAAATTAAATTCAAACCTAAATGAATGTCTAAATTTAAGGTAAAAGCAATTAATTCCTTCAAAATCATGGAAGTGGAAATTCCAG
TCAATAGCTTGTAATCACAAGCGCTAGACGTAACGAAAGTTCTTCAGTCAATGTAAGATAAAGAGGAAACGAATAAATAAAAAACTAAA
ATAACCGCTAAGATTAATCTGACTTGAAAGACTAAACTTTAAATCAATATGAAGATATCTTAAAAGAAAAGTAAAACATTTTGTAGCAT
CATTAAAATACAATGAATTAAACTCAAAGCAAAATGCAACCCTCATAATAACAAGTTATGATTAACAATTATACTTACCATACCAGTCA
AAACAATTTAAAAAATGTAAGCAGTAATTCATAATAATCTTAAAATATAAATAACTATCATTAAGTTAGTAACATTGAAAATAACATCA
ATAATGCAAATACATAGTAAATTTAAGAAAGACCTAAACAAAATTCTTAATAATAAAATATTGGCTACTAAACCACCCACAATAATCAT
AAAAGTATTAACATCTAATAATCATAAAATTAAAATATAAATAATTACAATTGCAATATTGAAAATAATATTAATAGTGCTAATTTGTA
ACCATTACTTGAAAAAAATAAATTTAATGTTTAATAATAACACTTTAACAGTAATACTCTGAACTCATATTCCTTTCTCAGCAACAACA
ATAATATTAGCAGCACATCTAATTTTTCTTCTTTTGGAACACTTCCAAAAGGCACATCAGCTAGTATTTTGAAATAATAATAATAATAA
TAACAATAACAATAAAACACCCAAACAACTATTAATTCTATGCTATTGCATTTATAGTAAAATGACTTATAAAAAAATTACTTTTTAAA
TTAATAAAAAATTTAAAATAGCAGCAACAACGGTTAGAATTAATATGCATATTAAAAGAATAATCAATTAGAAATCAATTCTGCTTCTA
ATTTGGCTCTAACAGATTAAGATGCTTTGAAAGAGTTACTTAGCAAAACTACTTCAACAAAGAGTGCAATCAAAAACGCTGCCAACTAC
TTCTCTTCTCAACAACATAAATAATAAGTTTAAAATAATATAAATCTTTAATAAGAAGAGAAACCTGGATTTTAATCTGAGCGTCTTAA
CAACAACAACAATAATACAAACAATACTTAATTTATTCTTTAAAACCAAGTTGCTTAAAAAAAAAATGAATGTATAAACAATTAAAATA
AAATCAATGTTCAATCTCACTTAAAGGTTTAAAATTAATAAAAAGCTTAAAACAATCCTAATAATTCTTAAGCAGTAGAACAAATGTAC
TAAAACTAAGCTCCCATCTAGAGAAAAATACAAGAAAAGATACAAAAATTTACATAAATATAAACATAAATTCAAAACTATAATCCAAT
CTAAACCTAAAATCTGACATAAAATTCTAATAAATCTCATTTAAAAGCAAACCCTATTTCTATATTGTCTTAGGTAAACTCGTATGGAA
TTGTAAACAATTCAAAGTAGCCAACACCAGCAATACCCTCAGTTTCTTCTGTTAGCAGTACTTAAAATCTTCATTAATAAAAAACTAAT
GAAACTCTTAGCAATTTTACTGTTTCTCCAAGTGTTCCAAGTACTTAAGTGCAAATTACAAATCAAATAGGATTATTGAGCTCTAATTC
AAATAATAGTACTAATAATAACAATATAAATAATAATTATTATAGTAATCTAAGTAACAAAGCCGAATATCCACATATTTAAAATAATT
AAATCGGAGTAAACAATATTAGTTAACTTGGATAGTTGTCTGGATAATGTTCTAGTAATAACAATAACACGACTCAAAATAGTGCCTTA
AATTAAAATAATCATAAAAATTTGCTTTCAACTGCTATAAAATCCGAAACTACTCAGGTTTCTAATTTTAGTTAAGATTCTCCTCTTGT
ATGTAATGAAAATCTATCAGGAATTCTAACAAACAGATATGGCGATAATTCATAATTTAAAAATTTACTATCCTGCAAAAGCAACAACA
TGTCAAGTAATAATATCAATCCAGGAACTTATAATAGCAACACTTCAAACAATATACACTAAAATTCATCATCTTTTACTTAAACTTTA
TCAGAAAACAAAGACAAAATACAAAATAATATTTAAATTTAAAATTATATTTCTACTTAACATTAAACCTCTCAAATTAATTCAAAGAT
TGGAATCACTAATATTTAATTAATTGAAGAATACGATAATTCGATTCCTTATAGTCAATCATAGTAATCTTAAATTTTAAATAATTATG
GTGCTTAGCCTAATAATGCTTCTAACAGTTAAAGCAATAATAATAATAATATTAATAATAGCAATTGTGGATTTAAACCTACTCATAAA
AAGAATTTAAGCAATCATCTTATAAATAAATTAGGAGCTATTTGCAAAAGTGCCACTGAAGGATCATAAACCTATACCTAAAGTTCTCA
AAATAACTCTTAAAACAAAGATTCTTTCAACAATAACAACAACACTTTAAGCAGTAATTAAAATAAAACTAAAAATAATACTGTTAGTA
TTAGTCTAAATAACAATAACACTAATAATCTTTAAAGTAATACTTTGAGTTAAGTCCAAGATTTAGATCTCAAAAAAGAAGATACTGTT
GTCACAAATTATGAGTTAGAAGTTGTGGAAGAAAGCTTAAAAGATAGCATTGAGCAATTTAGTACTAAATAAAATGTTTAAATCAACAA
AATACCAAATAACTTAAAATTAGATAAATAAAAAAATAAAGAATAAAAGGAAAGTCTTGAATAGCAGCTTTTAAATTTCTAGCATTAAA
ATATCTTAAGTGAGAATTTCGAATACAGAATTAAGGATATGAAAGAATAGGAAATAGACTATCTGCCAAATCCAGATTATTTTGATAAT
TAAACAGAAATTACTTGTATGATGAGATGCATTCTATTTGATTGGATGTTTGATGTTTGCATGAGCCTCATGCTAAAAAGAGAAACTGT
TTATCTTTCTTTAAATTATGTTGATAGGTATCTCAGCTAAAAATAGGTTACAAAGCTTAACCTTTAACTTGTTGGAGCTGTTTCTCTTT
ATATGGCTTGTAAAATAGAAGAAATTCAACCTCCTTCCATTTCAGAGTTTGCAAAATGTACTGATGATGGTTATACAGTAGCATAAATT
gtagaatatgaactgctaatgttaaaagtaattaatttattattaaaatatcttttcattcaaataaattttaattagcttatataaaa
taacaaaaataataaataccttacttacttattaattaattaattaattaataaagcgatttatctccaaaagttttttaaaatacaaa
tgaatttgtttggaggaagataaaaattaagaataaattagaaattacaataataattaagataaaaagcaattttttaaaaactttta
atttagatagttcaaaaaatatatatgaatatttaaaatatttttatttttaattattattaaataaataaaaataaattataataata
aatagGCTTTTGATTGGAAATTAAATCCACCAACATAAATAACTTGGTTGAACATGTATACTGAGATATGGGATAGATTCATAAAATCT
AGTTTTGATAAGCCAAATAAATATCAAATAAATTTTGTTTAAGATCAAAAGATTTCAAACAAGAATTACTTAATTCTCTACAGAATTCC
TGAATAGAAAAGTTACATGCTTTTTAGATAAATGGTGTAGATTTTGGATTTAATGTAATTAGATGCTAATGTATTAAGATTTGAAAGCA
GAATGCTAATTGCTTCATTAATGTACTTGTAGCTAGGCATTTATTATAAATAATTCACTAAAAAATAAGTTCATTCAGGATTCAATGAA
AAATACTTATTTGCTAATTCACTTAATAATTTCAACGAATACTTTAATTCTTTTTTGACTCAGCACTTCTAATTTAATCTGATTGACAT
ACTTTCTACTTTGCAACATGTAGCAACTTTCTTTGACGTAGAATATAATTATTCACTTTCTCCAGCAGCCTAAATGATTGATCCTACAA
ATAACGAGGTAAATATTATAATTCACTTTAAAAATATATAATTAAATAAATTTATTTATTTATTTCAATAGATGTCTTATGAAGAATAT
TTATCCTATTAAACTTATTTCCCTAATGGTATTGATTATATAAGGAAAAGATACTGCTCATGA
Figure 1: TTHERM_00332170 gene with introns (red) and primers (yellow)
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
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
RTPCR of TTHERM_00332170 gene expression during conjugation
1400
1200
band intensity
1000
800
Series1
600
400
200
0
1
V1 V22 S13 S24 0 5
S1 S2 0
V1 V2
14 11
15 16
18 15
19 20
1 6 27 38 59 610 711 812 13
9 10
12 17
13 14
16 21
17 22
18
1
2
3
4
5 6of conjugations
7 8
9 10
hous
11
12
13
14 15 16
17
18
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: http://www.ciliate.org/conjugation.shtml
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