Cell division in higher eukaryotes is mainly controlled by p34cdc2, a serine/
threonine protein kinase, and/or related kinases, and by other components of these
kinase complexes. I present evidence that CDC2-like kinases also occur in the ciliate
Paramecium tetraurelia. The protein encoded by the isolated Paramecium cdc2 homologue
did not bind to pl3sucl , was localized in the macronucleus, its associated kinase activity
was high at the initiation of macronuclear DNA synthesis, and it was active as a
monomer.
To study the relationship between the cellular and molecular events of cell cycle
regulation, synchronous cultures are essential. However, in Paramecium, the only reliable
technique for obtaining synchronous cell populations has been hand-selection of dividing
cells. This technique is only useful for small samples and impractical for biochemical
analysis. In this thesis, centrifugal ehxtriation, which fractionates the cell population on the
basis of sedimentation properties with minimal perturbation of metabolic function, was
applied to the ciliate Paramecium tetraurelia. Only the smallest cell fractions were well
synchronized and exhibited synchrony and cell cycle duration equivalent to hand-selected
samples. These small cell fractions consisted of a highly synchronous G1 cell population,
which was easily obtained by this technique and used for all subsequent molecular and
biochemical analysis.
With a combination of various polymerase chain reaction (PCR) techniques, a cdc2
homologous sequence was isolated from Paramecium which is referred to as cdc2PtA.
The genomic Paramecium cdc2PtA gene contained two short introns near the 5'-end. The
corresponding amino acid sequence exhibited about 50 % identity to the cdc2 proteins of
other eukaryotes. The Paramecium cdc2PtA gene-encoded protein was 11 amino acids
longer than that of Schizosaccharomyces pombe. It had most of the catalytic sites required
for CDC2 kinase activity, especially those phosphorylation sites which regulate
CDC2 kinase activity in other organisms. There was one amino acid change in the highly
conserved PSTAIRE region and other changes in regions which are required for interaction
with other regulatory proteins, especially the pl3*"e / binding sites. Southern blot
analysis as well as isolation of a second incomplete cDNA sequence from the 3'-end indicated
that Paramecium has multiple cdc2 genes.
Northern blotting results showed that the Paramecium cdc2PtA gene was much
more strongly expressed in actively dividing cells than in starved stationary phase cells in
which cdc2PtA mRNA was almost undetectable. There was no significant change in
cdc2PtA mRNA level throughout the vegetative cell cycle. Polyclonal antibodies were
produced against both a synthetic peptide from the C-tenninal region and a GSTCDC2PTA
fusion protein which contained a third of the Paramecium cdc2PtA protein
from the N-terminal region. Both antibodies recognized a 36 kDa polypeptide on Western
blots. The antibodies did not cross-react with protein extracts from Tetrahymena or S.
pombe, nor with the Paramecium 34 kDa polypeptide which was detected by anti-
PSTATJRE antibody. The Paramecium CDC2PTA protein level decreased slightly when
cells entered stationary phase and was invariant throughout the cell cycle, similar to its
transcription pattern. Indirect immunofluorescence results showed that Paramecium
CDC2PTA protein was located in the macronucleus, but not observed in the micronuclei
or cytoplasm Upon starvation, the strength of the fluorescence signal in the macronucleus
dropped slightly, consistent with the result from Western blotting.
Native Paramecium CDC2PTA kinase was immunoprecipitated with the Paramecium
CDC2PTA specific antibody. The precipitated CDC2PTA kinase phosphorylated
both bovine histone HI and casein in vitro, but not retinoblastoma (Rb) protein. Using
histone HI as substrate, CDC2PTA kinase activity was assayed in the ehitriation synchronized
samples. Histone HI kinase activity was high during the early stages of the cell cycle
and reached a peak at around 2.5 hr after ehitriation, which corresponded approximately
to the time of the initiation of macronuclear DNA synthesis. This suggests that the isolated
Paramecium CDC2PTA kinase may be associated with the regulation of macronuclear
DNA synthesis.
When Paramecium extracts were probed with anti-PSTAIRE antibody, two
polypeptides were detected. The major one migrated at 36 kDa was apparently recognized
by anti-CDC2PTA antibody. The minor one migrated at the same position as S. pombe
p34cde2 protein. Only the faster migrating one showed affinity for p 13™c7 protein. The
phosphotransferase activity of the p13sucl / precipitable protein was very low at early stages
and increased at around 1.5 hr before cell division. This kinase activity increase corresponded
to the point of commitment to division in Paramecium.
Immunoprecipitation results showed that Paramecium CDC2PTA kinase occurred
principally as monomers. This was further confirmed by glycerol density gradient centrifugation
and gel filtration. These monomers were active as a histone HI kinase in vitro.
These observations indicate that isolated Paramecium CDC2-like kinase differs from typical
CDC2 kinases in terms of interaction with and regulation by other cell cycle regulatory
components. / Science, Faculty of / Zoology, Department of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/6209 |
| Date | 05 1900 |
| Creators | Tang, Liren |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Format | 9634409 bytes, application/pdf |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
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