Biology Reference
In-Depth Information
FMNH
2
and light. The expression of
luxAB
as reporter gene ceases once cell death ensues. That is
why a promoterless
luxAB
was used as a reporter gene to give rise to transcriptional fusions with
the desired promoter of the gene to be expressed. The expression of the transcripts in response to
nitrogen step-down or various environmental shifts could be identifi ed with increased or decreased
luminescence. Since the plasmid used for the transfer lacks the restriction sites for AvaIII and AvaII
enzymes, it is not subjected to the degradative action of the corresponding enzymes inside
Anabaena
sp. strain PCC 7120 cells upon transfer (Elhai and Wolk, 1990; Wolk
et al
., 1991; Ernst
et al
., 1992;
Maldener
et al
., 1994; Cai and Wolk, 1997). Elhai and Wolk (1990) successfully demonstrated the
expression of large and small subunits of RuBisCO in all vegetative cells of
Anabaena
sp. strain PCC
7120 when
luxAB
was introduced downstream of P
rbcLS
. Likewise, the expression of
glnA
(GS gene)
was found in vegetative cells as well as heterocysts but the expression of nitrogenase genes
nifHDK
was restricted to heterocysts.
Anabaena
sp. PCC 7118, a heterocystless mutant strain, fi xed nitrogen
under anaerobic conditions and transcriptional fusions of
luxAB
with P
nifHDK
showed expression
in specialized cells that looked different (lacking phycocyanin and occurring at different lengths
all over the fi lament) from vegetative cells. These results emphasize that
Anabaena
sp. PCC 7118
though lacked full development of heterocysts has retained the pattern (Elhai and Wolk, 1990). A
Tn5 derivative, Tn5-1063 with
luxAB
as reporter gene has advantages of locating the sites of gene
insertions, mapping their position and subsequent cloning. By sequencing the identity of the gene
is known (Wolk
et al
., 1991).
The
gfp
gene of jellyfi sh
Aequoria victoria
, which encodes green fl uorescent protein (GFP), has
been cloned after two decades of its intial discovery. GFP as a reporter can be used in the hosts across
all kingdoms and different colour variants like blue, yellow and red can be used together in systems
to fi nd out interplay of genes and their products. Upon excitation by UV-light (395 nm) the inert
gfp
reporter gene emits green light (509 nm) that can be easily visualized by using epifl uorescence
microscopy. Due to its fairly longer half-life extending many bacterial generations it sometimes
creates problem as the dead cells also continue to show luminescence unlike
luxAB
reporter. In
view of this, construction of
gfp
variants with shorter half-lives of 40, 60 and 110 min was feasible
with the addition of degradative tag to the C-terminal end of wild-type
gfp
(Andersen
et al
., 1998).
The expression of a number of early genes after nitrogen step-down in
Anabaena
sp. strain PCC
7120 has been studied with the help of
gfp
transcriptional fusions (Khudyakov and Golden, 2004;
Wang and Xu, 2005; Zhao
et al
., 2005; Zhang
et al
., 2009; Mella-Herrera, 2010). Cell-type specifi c gene
expression in
N
.
punctiforme
ATCC 29133 was analysed by the construction of two transcriptional
shuttle vectors using
gfp
as the reporter gene. Fluorescence of GFP was mainly restricted to the
heterocysts when P
hetR
-
gfp
transcriptional fusion was introduced into
N
.
punctiforme
ATCC 29133
(Argueta
et al
., 2004).
9) GENES ESSENTIAL DURING DEVELOPMENT
A)
Early genes
:
Regulatory genes
i) ntcA
:
Chastain
et al
. (1990) identifi ed a DNA-binding factor (VF1) from
Anabaena
sp. strain PCC
7120 cells that binds to three adjacent sites in the
xisA
upstream region. Vega-Palas
et al
. (1990) fi rst
identifi ed
ntcA
gene (for nitrogen control) in
Synechococcus
sp. PCC 7120 while characterizing
pleiotropic mutants that were unable to utilize inorganic nitrogen in a form other than ammonium.
The gene product of
ntcA
is a DNA-binding protein and a transcriptional activator that belongs to
the CRP-family of bacterial transcriptional regulators (Vega-Palas
et al
., 1992). The DNA-binding
activity is mediated by the presence of a helix-turn-helix at its C-terminal end (Vega-Palas
et al
.,
