More than 100 bacterial isolates from various marine habitats were screened for AHL production by using gfp reporter constructs based on the lasR system of Pseudomonas aeruginosa and the luxR system of Vibrio fischeri. Of the 67 Alphaproteobacteria tested, most of which belonged into the so-called Roseobacter clade, 39 induced fluorescence in either one or both sensor strains up to 103-fold compared to controls. Acylated homoserine lactones were identified by GC-MS analysis and shown to have chain lengths of C8, C10, C13-C16, and C18. One or two double bonds were often present, while a keto or hydroxyl group occurred only rarely in the side chain. Most strains produced several different AHLs. C18-en-HSL and C18-dien-HSL were produced by Dinoroseobacter shibae, an aerobic anoxygenic phototrophic bacterium isolated from dinoflagellates, and are among the longest AHLs found to date. Z7-C14-en-HSL, which has previously been detected in Rhodobacter sphaeroides, was produced by Roseovarius tolerans and Jannaschia helgolandensis. This signal molecule was synthesised and shown to induce a similar response to the culture supernatant in the respective sensor strain. The widespread occurrence of quorum-sensing compounds in marine Alphaproteobacteria, both free-living strains and those associated to eukaryotic algae, points to a great importance of this signalling mechanism for the adaptation of the organisms to their widely different ecological niches.

We have isolated and determined DNA sequence for the 5'-flanking regions of three Arabidopsis thaliana polyubiquitin genes, UBQ3, UBQ10, and UBQ11. Comparison to cDNA sequences revealed the presence of an intron in the 5'-untranslated region at the same position immediately upstream of the initiator methionine codon in each of the three genes. An intron at this position is also present in two sunflower and two maize polyubiquitin genes. An intron is also found in the 5'-untranslated regions of several animal polyubiquitin genes, although the exact intron position is not conserved among them, and none are in the same position as those in the higher plant polyubiquitin genes. Chimeric genes containing the 5'-flanking regions of UBQ3, UBQ10, and UBQ11 in front of the coding regions for the reporter enzyme Escherichia coli beta-glucuronidase (GUS) were constructed. When introduced transiently into Arabidopsis leaves via microprojectile bombardment, all resulted in readily detectable levels of GUS activity that were quantitatively similar. The introns of UBQ3 and UBQ10 in the corresponding promoter fragments were removed by replacement with flanking cDNA sequences and chimeric genes constructed. These constructs resulted in 2.5- to 3-fold lower levels of marker enzyme activity after transient introduction into Arabidopsis leaves. The UBQ10 promoter without the 5' intron placed upstream of firefly luciferase (LUX) resulted in an average of 3-fold lower LUX activity than from an equivalent construct with the UBQ10 intron. A UBQ3 promoter cassette was constructed for the constitutive expression of open reading frames in dicot plants and it produced readily detectable levels of GUS activity in transient assays.

Genome-wide expression signatures detect specific perturbations in developmental programs and contribute to functional resolution of key regulatory networks. In maize (Zea mays) inflorescences, mutations in the RAMOSA (RA) genes affect the determinacy of axillary meristems and thus alter branching patterns, an important agronomic trait. In this work, we developed and tested a framework for analysis of tag-based, digital gene expression profiles using Illumina's high-throughput sequencing technology and the newly assembled B73 maize reference genome. We also used a mutation in the RA3 gene to identify putative expression signatures specific to stem cell fate in axillary meristem determinacy. The RA3 gene encodes a trehalose-6-phosphate phosphatase and may act at the interface between developmental and metabolic processes. Deep sequencing of digital gene expression libraries, representing three biological replicate ear samples from wild-type and ra3 plants, generated 27 million 20-to 21-nucleotide reads with frequencies spanning 4 orders of magnitude. Unique sequence tags were anchored to 3#-ends of individual transcripts by DpnII and NlaIII digests, which were multiplexed during sequencing. We mapped 86% of nonredundant signature tags to the maize genome, which associated with 37,117 gene models and unannotated regions of expression. In total, 66% of genes were detected by at least nine reads in immature maize ears. We used comparative genomics to leverage existing information from Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) in functional analyses of differentially expressed maize genes. Results from this study provide a basis for the analysis of short-read expression data in maize and resolved specific expression signatures that will help define mechanisms of action for the RA3 gene.