G. Differential Gene Expression
1.) DECAL Library
Cyanobacteria utilize a number of regulatory strategies that enable them to
optimize their survival in restricted growth conditions, including global changes
in gene expression. Synechocystis sp. Strain PCC 6803, a unicellular,
transformable heterotrophic cyanobacterium, has become a particularly valuable
model organism now that its genome has been completely sequenced. It is the
first cyanobacterium to be sequenced, but more will follow shortly. Thus, the
ability to successfully prepare microarrays of the Synechocystis genome
is of value to many researchers interested in photosynthesis, nitrogen fixation
and environmental perturbations.
Various techniques, such as subtractive cDNA cloning, differential display
and DNA micro-arrays, have been used to analyze global gene expression under
different growth and environmental conditions. These procedures are easier to
perform in eukaryotes, due to the presence of a poly(A) tail on mRNA that permits
easy separation from rRNA. Unfortunately, bacterial mRNA lacks this poly(A)
tail and can not be routinely extracted from total RNA. This problem also makes
it difficult to use microarrays in bacteria, because of the high background
that is observed when total RNA is used as a labeled probe. One way to study
the differential expression in prokaryotic organisms with small genomes is to
use membranes on which are spotted arrays of Escherichia coli cells
containing a library of clones. Nonetheless, an absolute requirement when using
such membranes is the removal of the abundant rRNA from total RNA before labeling
to avoid high background that may interfere with signal analysis. A method called
Differential Expression using Customized Amplification
Library (DECAL) has been efficiently used in global comparisons of gene
expression in Mycobacterium tuberculosis (Alland et al, 1998). DECAL
accomplishes this by first creating a customized amplification library (CAL)
of genomic DNA that has been manipulated for optimal performance during analysis.
The success of CAL depends on three factors: (i), physical removal of abundant
sequences, i.e, rRNA genes; (ii), reduction in the complexity of the sequences
and conversion of all DNA sequences into fragments of smaller and similar size;
and (iii), selection of sequences that amplify efficiently.
DECAL Paper
We constructed such a DECAL for Synechocystis sp. PCC 6803. To
examine proof-of-concept, the DECAL was used to identify the differentially-expressed
genes in iron-deficient vs. iron-reconstituting cells of Synechocystis
sp. strain PCC 6803. A number of genes were identified, such as isiA, idiA,
psbA, cpcG and slr0374, whose expression either increased or decreased
in response to iron-availability. Further analysis led to the identification
of additional genes (e.g., psbC, psbO, psaA, apcABC, cpcBAC1C2D and
nblA) that were differentially regulated by iron availability. Expression
of cpcG, psbC, psbO, psaA, apcABC and cpcBAC1C2D increased,
whereas that of isiA, idiA, nblA, psbA and slr0374 decreased
in iron-reconstituting cells. S1 nuclease protection studies showed that increased
transcript levels of psbA in iron-deficient cells was due to the increased
expression of both psbA2 and psbA3 genes, although the steady-state
level of psbA2 remained higher as compared to that of psbA3.
This study demonstrates that DECAL can be applied to study the differential
gene expression in Synechocystis sp. strain PCC 6803 under altered
environmental conditions.
The use of the DECAL gave us valuable experience, but this approach has limitations.
The library is based on arrays of cosmid clones. The advantage to this system
was the requirement for fewer clones to prepare the array, a feature of particular
importance before the establishment of our Agricultural Genomics Center with
the aid of an NSF grant. However, the clones carry rather large fragments (35-45kb)
and it is possible that an individual clone contains some genes which are down-regulated
and some which are up-regulated. Such clones would show little or no change
between two conditions. We recognized this deficiency at the initiation of this
project, but it was a good way to begin such studies. We have now constructed
a 6,000 clone library based on 2kb fragments that is being used to continue
our studies. We then graduated to the use of microarrays in which each of
the 3,168 genes in Synechocystis sp. PCC 6803 can be studied as discrete
entities.
2. Genomic Microarray of Synechocystis sp. PCC 6803
DNA microarray technology can provide an efficient and cost-effective means
to help define the functions and functional interrelationships among the numerous
proteins identified by large-scale genomic sequencing efforts. The focus
of our project was to develop DNA microarray technology for the unicellular cyanobacterium,
Synechocystis sp. PCC6803. A determination of differential transcriptional profiles associated
with environmental stress regimes will be critical for a better understanding
of how autotrophs optimize energy metabolism when challenged with environmental
stress. The described approach for microarray analysis involves: 1) utilization
of genomic sequence data to generate a set of gene-specific PCR products; 2)
immobilization of the resultant DNA fragments in array format; and, 3) hybridization
of the array with labeled RNA from control and treated samples to reveal treatment-induced
differences in the abundance of the specific mRNAs that correspond to the immobilized
genes.
Among the cyanobacteria, Synechocystis sp. Strain PCC 6803 has proven
to be one of the best model organisms. It is a unicellular organism that is
naturally transformable [6, 22] and capable of photoautotrophic and heterotrophic
growth in the absence of light (when a carbon source such as glucose is provided).
This later property permits analysis of photosynthetic mutants in Photosystem
I and II and this attribute led to the decision to make Synechocystis
the first cyanobacterium for which the entire genome would be sequenced (for
details see web site http://www.kazusa.or.jp/cyano/cyano.html) [11]. In addition,
the ability to make knockout mutations in virtually any gene and the relative
ease of protein analysis makes this strain among the most valuable of the prokaryotes
for functional genomics.
When the sequence was first completed, 3168 potential proteins were identified,
of which over 50% could not be compared to known proteins [13]. Many of the
genes coding for metabolic functions could be categorized, and Synechocystis
has already been a valuable resource for the study of photosynthesis, respiration
and the biosynthesis of prosthetic groups and the like. At the same time, cyanobacteria
acclimate to changing environments using two-component regulatory systems and
80 genes for two-component signal transducers have been identified [12]. Even
more importantly, researchers in many areas of plant biology are utilizing the
Synechocystis database to search for proteins of interest. This especially
includes proteins involved in chloroplast structure and functions: e.g., chloroplast
envelope proteins, protein translocation systems, phytochromes, vitamin synthesis
and ion uptake. This microarray will be invaluable for analysis of certain physiological
studies in chloroplasts and will thus simplify gene regulation analysis in plants.
This is such a rich mine to be tapped that these efforts will continue for some
time. The interest of the broader plant community is one reason that we have developed a relatively inexpensive and reproducible supply of microarrays.
The experience gained with the Synechocystis microarray will also be
of value once other cyanobacterial genomes are sequenced and when other natural
strains are collected and analyzed for environmental adaptations. Since microarray
analysis does not require that the genome be sequenced, it is highly likely
that cyanobacteria, algae and related aquatic organisms will be isolated for
analysis of physiological and metabolic characteristics and their response to
environmental perturbations. The Synechocystis microarray will be a
valuable adjunct for all such studies.
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