BackgroundThe size and complexity of conifer genomes has, until now, prevented full genome sequencing and assembly. The large research community and economic importance of loblolly pine, Pinus taeda L., made it an early candidate for reference sequence determination.ResultsWe develop a novel strategy to sequence the genome of loblolly pine that combines unique aspects of pine reproductive biology and genome assembly methodology. We use a whole genome shotgun approach relying primarily on next generation sequence generated from a single haploid seed megagametophyte from a loblolly pine tree, 20-1010, that has been used in industrial forest tree breeding. The resulting sequence and assembly was used to generate a draft genome spanning 23.2 Gbp and containing 20.1 Gbp with an N50 scaffold size of 66.9 kbp, making it a significant improvement over available conifer genomes. The long scaffold lengths allow the annotation of 50,172 gene models with intron lengths averaging over 2.7 kbp and sometimes exceeding 100 kbp in length. Analysis of orthologous gene sets identifies gene families that may be unique to conifers. We further characterize and expand the existing repeat library based on the de novo analysis of the repetitive content, estimated to encompass 82% of the genome.ConclusionsIn addition to its value as a resource for researchers and breeders, the loblolly pine genome sequence and assembly reported here demonstrates a novel approach to sequencing the large and complex genomes of this important group of plants that can now be widely applied.

Renilka reniformis is an anthozoan coelenterate capable of exhibiting bioluminescence. Bioluminescence in ReniUa results from the oxidation of coelenterate luciferin (coelenterazine) by luciferase [Renilka-luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.51. In vivo, the excited state luciferin4uciferase complex undergoes the process of nonradiative energy transfer to an accessory protein, green fluorescent protein, which results in green bioluminescence. In vitro, Renila luciferase emits blue light in the absence ofany green fluorescent protein. A Renifa cDNA library has been constructed in Agtll and screened by plaque hybridization with two oligonucleotide probes. We report here the isolation and characterization of a luciferase cDNA and its gene product. The recombinant luciferase expressed in Escherichia coli is identical to native luciferase as determined by SDS/PAGE, immunoblot analysis, and bioluminescence emission characteristics.Renilla reniformis (class Anthozoa) is a bioluminescent soft coral found in shallow coastal waters ofNorth America, which displays blue-green bioluminescence upon mechanical stimulation (1, 2). The components involved in Renilla bioluminescence have been described in detail (3). Renilla luciferase [Renilla-luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.5] catalyzes the oxidative decarboxylation of coelenterazine in the presence of dissolved oxygen to yield oxyluciferin, C02, and blue light (Am. = 480 nm) (4). This reaction has a bioluminescence quantum yield of =7%. The stoichiometry of this reaction and the detailed mechanism leading to excited-state formation have been described (4,5).The color of in vitro-catalyzed bioluminescence changes from blue to green upon addition of submicromolar amounts of an energy-transfer acceptor green fluorescent protein (GFP), which has been purified from Renilla and characterized (6). This green fluorescence (Amax = 509 nm) is identical to the in vivo emission in Renilla. The energy-transfer process is nonradiative; an increase in both the quantum yield (6) and calculated lifetimes has been determined for this process (7). Luciferase and GFP form a specific 1:1 rapid equilibrium complex in solution (8).The elucidation of mechanisms involved in nonradiative energy transfer processes as well as determination of detailed structural information on both luciferase and GFP have been hindered by a lack of material. To overcome this, we have cloned, sequenced, and expressed in Escherichia coli a cDNA encoding Renilla luciferase. § MATERIALS AND METHODS Amino Acid Sequence Determination of ReniUa Luciferase. Native Renilla luciferase was isolated as described (4). Purified luciferase was digested with Staphlococcal protease V-8 (9). The resulting peptides were purified by HPLC and subjected to NH2-terminal Edman sequencing as described (10). Based on these peptide sequences two 17-base oligonucleotide probes were synthesized with an Applied Biosystems DNA synthesizer at the Molecular Genetics Instrumentation Faci...

The infectious yeast Candida albicans progresses through two developmental programs which involve differential gene expression, the bud-hypha transition and high-frequency phenotypic switching. To understand how differentially expressed genes are regulated in this organism, the promoters of phase-specific genes must be functionally characterized, and a bioluminescent reporter system would facilitate such characterization. However, C. albicans has adopted a nontraditional codon strategy that involves a tRNA with a CAG anticodon to decode the codon CUG as serine rather than leucine. Since the luciferase gene of the sea pansy Renilla reniformis contains no CUGs, we have used it to develop a highly sensitive bioluminescent reporter system for C. albicans. When fused to the galactose-inducible promoter of GAL1, luciferase activity is inducible; when fused to the constitutive EF1␣2 promoter, luciferase activity is constitutive; and when fused to the promoter of the white-phase-specific gene WH11 or the opaque-phase-specific gene OP4, luciferase activity is phase specific. The Renilla luciferase system can, therefore, be used as a bioluminescent reporter to analyze the strength and developmental regulation of C. albicans promoters.Reporter genes which code for bioluminescent gene products, like the luciferases, have provided a very rapid method for analyzing the regulation of gene expression (4) and a highly sensitive method for single-cell analysis (38). Recently, we used the firefly luciferase gene (FLUC) fused in frame with the phase-regulated WH11 gene of Candida albicans as a reporter to functionally characterize the 5Ј upstream regulatory region of WH11 (29), but the analyses were restricted to Northern (RNA) blots because we were unable to identify a translation product of the firefly luciferase, either through enzyme activity or as a FLUC-related peptide in Western blots (immunoblots; unpublished observations). The lack of a detectable translation product was most likely due to a nontraditional codon strategy adopted by C. albicans and related species (19,22,23). These organisms use a tRNA with a CAG anticodon to decode the codon CUG as serine, while most organisms use CAG to decode the codon CUG as leucine. Recently, it was demonstrated that the traditional leucine isoacceptor tRNA for CUG from Saccharomyces cerevisiae is toxic to C. albicans (13). Furthermore, direct determination of the amino acid sequences of peptides derived from three aspartyl proteinases of C. albicans confirmed the presence of serine instead of leucine at nucleotide positions containing the CUG codon (38). FLUC contains nine in-frame CUG codons, making it highly unlikely that a functional luciferase could be expressed in C. albicans. In order to circumvent this codon problem, we have developed a reporter system for C. albicans using the luciferase gene RLUC of the sea pansy Renilla reniformis, which contains no CUG codons in its open reading frame (ORF). We have fused the Renilla luciferase gene to a number of promoters of C. albicans a...

A gene (yacK) encoding a putative multicopper oxidase (MCO) was cloned from Escherichia coli, and the expressed enzyme was demonstrated to exhibit phenoloxidase and ferroxidase activities. The purified protein contained six copper atoms per polypeptide chain and displayed optical and electron paramagnetic resonance (EPR) spectra consistent with the presence of type 1, type 2, and type 3 copper centers. The strong optical A 610 (⌭ 610 ‫؍‬ 10,890 M ؊1 cm ؊1) and copper stoichiometry were taken as evidence that, similar to ceruloplasmin, the enzyme likely contains multiple type 1 copper centers. The addition of copper led to immediate and reversible changes in the optical and EPR spectra of the protein, as well as decreased thermal stability of the enzyme. Copper addition also stimulated both the phenoloxidase and ferroxidase activities of the enzyme, but the other metals tested had no effect. In the presence of added copper, the enzyme displayed significant activity against two of the phenolate siderophores utilized by E. coli for iron uptake, 2,3-dihydroxybenzoate and enterobactin, as well as 3-hydroxyanthranilate, an iron siderophore utilized by Saccharomyces cerevisiae. Oxidation of enterobactin produced a colored precipitate suggestive of the polymerization reactions that characterize microbial melanization processes. As oxidation should render the phenolate siderophores incapable of binding iron, yacK MCO activity could influence levels of free iron in the periplasm in response to copper concentration. This mechanism may explain, in part, how yacK MCO moderates the sensitivity of E. coli to copper.

Serial analysis of gene expression was used to profile transcript levels in Arabidopsis roots and assess their responses to 2,4,6-trinitrotoluene (TNT) exposure. SAGE libraries representing control and TNT-exposed seedling root transcripts were constructed, and each was sequenced to a depth of roughly 32,000 tags. More than 19,000 unique tags were identified overall. The second most highly induced tag (27-fold increase) represented a glutathione S-transferase. Cytochrome P450 enzymes, as well as an ABC transporter and a probable nitroreductase, were highly induced by TNT exposure. Analyses also revealed an oxidative stress response upon TNT exposure. Although some increases were anticipated in light of current models for xenobiotic metabolism in plants, evidence for unsuspected conjugation pathways was also noted. Identifying transcriptome-level responses to TNT exposure will better define the metabolic pathways plants use to detoxify this xenobiotic compound, which should help improve phytoremediation strategies directed at TNT and other nitroaromatic compounds.