Biology Reference
In-Depth Information
gene in
Thermosynechococcus elongatus
BP-1, a thermophilic cyanobacterium. Rhythmic expression
of the gene, as measured by the luminescence with an automated monitoring apparatus, occurred
with a period close to 25 h for at least 10 days in constant light and temperature. The rhythm was
temperature compensated over a wide range of temperatures (30 to 60ºC) (Onai
et al
., 2004).
III. CIRCADIAN CLOCK
Three essential components have been identifi ed, i.e. the circadian clock also known as the oscillator,
a photoreceptor for setting the phase of the clock and a receptor for perceiving environmental
stimuli such as temperature (input pathway) and a means of relaying clock information to the
various behaviours that are under circadian control (output pathway). The bioluminescence system
with reporter
luxAB
marker developed for S.
elongatus
PCC 7942 fulfi lls all these requirements for
explaining cyanobacterial circadian clock. Knowledge that has been generated on the circadian
clock in cyanobacteria can be useful to understand circadian clocks in higher organisms, including
mammals (Ditty
et al
., 2003; Mackey
et al
., 2008; Eguchi
et al
., 2008; Johnson
et al
., 2008a,b;
Johnson, 2010).
A)
The oscillator in
Synechococcus
:
The oscillator should be able to control rhythmicity and
period of all downstream behaviour. In order to examine this, mutants affected in clock phenotype
would be best suited for the purpose. Realizing this, Kondo
et al
. (1994) isolated mutants of strain
AMC149 after mutagenesis with methylmethane sulfonate and screened over 1,50,000 clones for
their bioluminescence. Twelve mutants were further characterized with altered bioluminescence
rhythms whose periods ranged from 16 h to 60 h. Certain of the mutants exhibited bioluminescence
at such a low amplitude that it can even be considered as having no rhythms (arrhythmia). They
also demonstrated that it is possible to clone mutant genes by complementation involving single
recombination strategy similar to that used for random insertion of
luxAB
genes throughout the
genome. In this regard, they identifi ed a period extender gene (
pex
) in
S
.
elongatus
PCC 7942 that has
a 22-h period and designated it as SP22. Kutsuna
et al
. (1998) showed by sequence analysis that SP22
did not have a mutation in the genomic DNA segment carried on pS1K1 (and that
sp22
mutation was
later found in a recently cloned new clock gene,
KaiC).
As a matter of fact,
pex
gene that was carried
on
pS1K1
was a suppressor gene for the
sp22
mutation.
Pex
gene encodes a protein of 148 amino acid
residues that is thought to modulate function of the central clock oscillator. Genetic analysis of such
mutants offered the possibility that the circadian clock (oscillator) in
Synechococcus
is under the control
of three ORFs that may form one operon. X-ray crystallography and biochemical characterization of
Pex from
S
.
elongatus
PCC 7942 revealed that the molecule has a α+β structure with a winged helix
motif and is expected to function as a dimer. By the winged portion, Pex is able to recognize dsDNA.
The DNA-binding ability of Pex has further been substantiated by the isolation of a
pex
mutant in
which Pex fails to bind to DNA. Due to this, the period-extension activity of
pex
gene is lost in the
mutant. So Pex is suggested to be a DNA-binding transcription factor (Arita
et al
., 2007).
B)
Genes that regulate the clock
:
Kondo
et al
. (1994) identifi ed the above three ORFs by screening
as many as 500,000 clones of
S
.
elongatus
PCC 7942 obtained after mutagenesis of AMC149 strain
by EMS and characterized at least 100 mutants with altered circadian phenotypes. These mutants
did not show any other altered phenotype except for the differences in the period of the rhythms.
The recognition of the mutants and their isolation was possible because of the addition of wild-
type
Synechococcus
DNA. The DNA retrieved from such mutants showed that the wild-type DNA
could complement the mutant phenotype. From these studies they concluded that a cluster of three
