Nuclear dynamics in higher plants.
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Cell Nucleus↗
Biomedical subjects
David W Galbraith
Publications and source records attributed to David W Galbraith.
A gene expression map of the Arabidopsis root.
A global map of gene expression within an organ can identify genes with coordinated expression in localized domains, thereby relating gene activity to cell fate and tissue specialization. Here, we present localization of expression of more than 22,000 genes in the Arabidopsis root. Gene expression was mapped to 15 different zones of the root that correspond to cell types and tissues at progressive developmental stages. Patterns of gene expression traverse traditional anatomical boundaries and show cassettes of hormonal response. Chromosomal clustering defined some coregulated genes. This expression map correlates groups of genes to specific cell fates and should serve to guide reverse genetics.
Arabidopsis↗
The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy.
BACKGROUND: DNA microarrays are a powerful tool with important applications such as global gene expression profiling. Construction of bacterial DNA microarrays from genomic sequence data using a two-stage PCR amplification approach for the production of arrayed DNA is attractive because it allows, in principal, the continued re-amplification of DNA fragments and facilitates further utilization of the DNA fragments for additional uses (e.g. over-expression of protein). We describe the successful construction and use of DNA microarrays by the two-stage amplification approach and discuss the technical challenges that were met and resolved during the project. RESULTS: Chimeric primers that contained both gene-specific and shared, universal sequence allowed the two-stage amplification of the 3,168 genes identified on the genome of Synechocystis sp. PCC6803, an important prokaryotic model organism for the study of oxygenic photosynthesis. The gene-specific component of the primer was of variable length to maintain uniform annealing temperatures during the 1st round of PCR synthesis, and situated to preserve full-length ORFs. Genes were truncated at 2 kb for efficient amplification, so that about 92% of the PCR fragments were full-length genes. The two-stage amplification had the additional advantage of normalizing the yield of PCR products and this improved the uniformity of DNA features robotically deposited onto the microarray surface. We also describe the techniques utilized to optimize hybridization conditions and signal-to-noise ratio of the transcription profile. The inter-lab transportability was demonstrated by the virtual error-free amplification of the entire genome complement of 3,168 genes using the universal primers in partner labs. The printed slides have been successfully used to identify differentially expressed genes in response to a number of environmental conditions, including salt stress. CONCLUSIONS: The technique detailed here minimizes the cost and effort to replicate a PCR-generated DNA gene fragment library and facilitates several downstream processes (e.g. directional cloning of fragments and gene expression as affinity-tagged fusion proteins) beyond the primary objective of producing DNA microarrays for global gene expression profiling.
Cyanobacteria↗
Temporal progression of gene expression responses to salt shock in maize roots.
Using a cDNA microarray containing 7943 ESTs, the behavior of the maize root transcriptome has been monitored in a time course for 72 h after imposition of salinity stress (150 mM NaCI). Under these conditions, root sodium amounts increased faster than in leaves, and root potassium decreased significantly. Although the overall free amino acid concentration was not affected, amino acid composition was changed with proline and asparagine increasing. Microarray analysis identified 916 ESTs representing genes whose steady-state RNA levels were significantly altered at various time points, corresponding to 11% of the ESTs printed. The response of the transcriptome to sub-lethal salt stress was rapid and transient, leading to a burst of changes at the three-hour time point. The salt-regulated ESTs represented 472 tentatively unique genes (TUGs), which, based on functional category analysis, are involved in a broad range of cellular and biochemical activities, prominent amongst which were transport and signal transduction pathways. Clustering of regulated transcripts based on the timing and duration of changes suggests a structured succession of induction and repression for salt responsive genes in multiple signal and response cascades. Within this framework, 16 signaling molecules, including six protein kinases, two protein phosphatases and eight transcription factors, were regulated with distinct expression patterns by high salinity.
Amino Acids↗
The sfr6 mutant of Arabidopsis is defective in transcriptional activation via CBF/DREB1 and DREB2 and shows sensitivity to osmotic stress.
The sfr6 mutant of Arabidopsis displays a deficit in freezing tolerance after cold acclimation. We previously observed that the transcripts of three cold-, ABA- and drought-inducible genes, each having a C-repeat motif or the drought-responsive element (CRT/DRE) in its promoter, failed to normally accumulate in this mutant. We now report that the effects of sfr6 upon transcript levels are reflected in the levels of the encoded proteins, confirming that the cold-inducible protein expression is affected by the sfr6 mutation. Using microarray analysis, we found not only that this effect may be general to cold-inducible genes with CRT/DRE promoter elements, but also that it extends to some other genes whose promoters lack a CRT/DRE element. The role of the CRT/DRE has been empirically tested by use of a synthetic promoter, confirming that the CRT/DRE is sufficient to confer the sfr6 effect upon expression. Tolerance of osmotic stress was also found to be reduced in sfr6, consistent with a role in osmotic stress tolerance for the cold-, ABA- and drought-inducible genes whose expression is affected by the sfr6 mutation.
Abscisic Acid↗
Comparison of RNA expression profiles based on maize expressed sequence tag frequency analysis and micro-array hybridization.
Assembly of 73,000 expressed sequence tags (ESTs) representing multiple organs and developmental stages of maize (Zea mays) identified approximately 22,000 tentative unique genes (TUGs) at the criterion of 95% identity. Based on sequence similarity, overlap between any two of nine libraries with more than 3,000 ESTs ranged from 4% to 20% of the constituent TUGs. The most abundant ESTs were recovered from only one or a minority of the libraries, and only 26 EST contigs had members from all nine EST sets (presumably representing ubiquitously expressed genes). For several examples, ESTs for different members of gene families were detected in distinct organs. To study this further, two types of micro-array slides were fabricated, one containing 5,534 ESTs from 10- to 14-d-old endosperm, and the other 4,844 ESTs from immature ear, estimated to represent about 2,800 and 2,500 unique genes, respectively. Each array type was hybridized with fluorescent cDNA targets prepared from endosperm and immature ear poly(A(+)) RNA. Although the 10- to 14-d-old postpollination endosperm TUGs showed only 12% overlap with immature ear TUGs, endosperm target hybridized with 94% of the ear TUGs, and ear target hybridized with 57% of the endosperm TUGs. Incomplete EST sampling of low-abundance transcripts contributes to an underestimate of shared gene expression profiles. Reassembly of ESTs at the criterion of 90% identity suggests how cross hybridization among gene family members can overestimate the overlap in genes expressed in micro-array hybridization experiments.
Contig Mapping↗
Monitoring large-scale changes in transcript abundance in drought- and salt-stressed barley.
Responses to drought and salinity in barley (Hordeum vulgare L. cv. Tokak) were monitored by microarray hybridization of 1463 DNA elements derived from cDNA libraries of 6 and 10 h drought-stressed plants. Functional identities indicated that many cDNAs in these libraries were associated with drought stress. About 38% of the transcripts were novel and functionally unknown. Hybridization experiments were analyzed for drought- and salinity-regulated sequences, with significant changes defined as a deviation from the control exceeding 2.5-fold. Responses of transcripts showed stress-dependent expression patterns and time courses. Nearly 15% of all transcripts were either up- or down-regulated under drought stress, while NaCl led to a change in 5% of the transcripts (24 h, 150 mM NaCl). Transcripts that showed significant up-regulation under drought stress are exemplified by jasmonate-responsive, metallothionein-like, late-embryogenesis-abundant (LEA) and ABA-responsive proteins. Most drastic down-regulation in a category was observed for photosynthesis-related functions. Up-regulation under both drought and salt stress was restricted to ESTs for metallothionein-like and LEA proteins, while increases in ubiquitin-related transcripts characterized salt stress. A number of functionally unknown transcripts from cDNA libraries of drought-stressed plants showed up-regulation by drought but down-regulation by salt stress, documenting how precisely transcript profiles report different growth conditions and environments.
Disasters↗