Synechococcus elongatus PCC 7942 is a model organism used to study the circadian
rhythm, a process that is driven by an endogenous biological clock that can be modulated
by external cues such as light and temperature. Some proteins have been identified that
are involved in circadian signal transduction in S. elongatus. Of them, KaiA, KaiB and
KaiC comprise the central oscillator components, which are essential for internal
timekeeping. SasA is an important protein in the output pathway, which passes the
information from central oscillator to downstream components, and thus controls
metabolic and behavioral processes. CikA is a major component in the input pathway,
which maintains synchrony of the oscillator with the environment. CikA is an unusual
phytochrome-like histidine protein kinase. It has a pseudo receiver domain which can not
accept a phosphoryl group. CikA is thought to be located at the poles of the cell through
interaction between PsR and some protein or protein complex that is also localized at the
poles. One of the potential CikA-interacting proteins identified through a yeast two
hybrid screen is called hypothetical protein 1. It specifically recognizes a PsR bait in a
yeast two hybrid assay. A bioinformatics analysis showed that there are predicted signal
peptide and transmembrane domains at the N-terminal and a cytochrome C homolog
domain at the C-terminal of Hyp1. Elucidating the features and function of Hyp1 will
provide us with useful information to understand the function and working mechanism of
CikA, and therefore will help us to clarify the signal transduction in the clock. In this
research, I used genetic, cell biological and biochemical approaches to study the features
and function of this newly identified clock component Hyp1.
To confirm the interaction between PsR and Hyp1 and complement the yeast two
hybrid data, I truncated Hyp1 (Thyp1) and purified soluble Thyp1. At the same time, I
obtained purified PsR. I tried to copurify the PsR and 6-histidine-tagged Hyp1 on a nickel affinity column. However, PsR non-specifically bound to the column, which eliminated
the utility of this approach to study their interaction.
In addition to using a biochemical approach to study Hyp1, I constructed three
hyp1 overexpression alleles for genetic analysis and two hyp1-yfp overexpression fusion
alleles for subcellular localization studies. All of them will help us to understand the
features and function of Hyp1.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/5850 |
| Date | 17 September 2007 |
| Creators | Guo, Haitao |
| Contributors | Golden, Susan S. |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Thesis, text |
| Format | 1186748 bytes, electronic, application/pdf, born digital |
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