DANPY (dimethylaminonaphthylpyridinium): an economical and biocompatible fluorophore.

Dyes with nonlinear optical (NLO) properties enable new imaging techniques and photonic systems. We have developed a dye (DANPY-1) for photonics applications in biological substrates such as nucleic acids; however, the design specification also enables it to be used for visualizing biomolecules. It is a prototype dye demonstrating a water-soluble, NLO-active fluorophore with high photostability, a large Stokes shift, and a favorable toxicity profile. A practical and scalable synthetic route to DANPY salts has been optimized featuring: (1) convergent Pd-catalyzed Suzuki coupling with pyridine 4-boronic acid, (2) site-selective pyridyl N-methylation, and (3) direct recovery of crystalline intermediates without chromatography. We characterize the optical properties, biocompatibility, and biological staining behavior of DANPY-1. In addition to stability and solubility across a range of polar media, the DANPY-1 chromophore shows a first hyperpolarizability similar to common NLO dyes such as Disperse Red 1 and DAST, a large two-photon absorption cross section for its size, substantial affinity to nucleic acids in vitro, an ability to stain a variety of cellular components, and strong sensitivity of its fluorescence properties to its dielectric environment.

Genome Sequences of Eight Bacterial Species Found in Coculture with the Haptophyte Chrysochromulina tobin.

The microalgal division Haptophyta uses a range of nutritional sourcing, including mixotrophy. The genome of a member of this taxon, Chrysochromulina tobin, suggests that interactions with its bacterial cohort are critical for C. tobin physiology. Here, we report the genomes of eight bacterial species in coculture with C. tobin.

Genome Sequence and Transcriptome Analyses of Chrysochromulina tobin: Metabolic Tools for Enhanced Algal Fitness in the Prominent Order Prymnesiales (Haptophyceae).

Haptophytes are recognized as seminal players in aquatic ecosystem function. These algae are important in global carbon sequestration, form destructive harmful blooms, and given their rich fatty acid content, serve as a highly nutritive food source to a broad range of eco-cohorts. Haptophyte dominance in both fresh and marine waters is supported by the mixotrophic nature of many taxa. Despite their importance the nuclear genome sequence of only one haptophyte, Emiliania huxleyi (Isochrysidales), is available. Here we report the draft genome sequence of Chrysochromulina tobin (Prymnesiales), and transcriptome data collected at seven time points over a 24-hour light/dark cycle. The nuclear genome of C. tobin is small (59 Mb), compact (∼ 40% of the genome is protein coding) and encodes approximately 16,777 genes. Genes important to fatty acid synthesis, modification, and catabolism show distinct patterns of expression when monitored over the circadian photoperiod. The C. tobin genome harbors the first hybrid polyketide synthase/non-ribosomal peptide synthase gene complex reported for an algal species, and encodes potential anti-microbial peptides and proteins involved in multidrug and toxic compound extrusion. A new haptophyte xanthorhodopsin was also identified, together with two "red" RuBisCO activases that are shared across many algal lineages. The Chrysochromulina tobin genome sequence provides new information on the evolutionary history, ecology and economic importance of haptophytes.

The mitochondrial and chloroplast genomes of the haptophyte Chrysochromulina tobin contain unique repeat structures and gene profiles.

BACKGROUND: Haptophytes are widely and abundantly distributed in both marine and freshwater ecosystems. Few genomic analyses of representatives within this taxon have been reported, despite their early evolutionary origins and their prominent role in global carbon fixation. RESULTS: The complete mitochondrial and chloroplast genome sequences of the haptophyte Chrysochromulina tobin (Prymnesiales) provide insight into the architecture and gene content of haptophyte organellar genomes. The mitochondrial genome (~34 kb) encodes 21 protein coding genes and contains a complex, 9 kb tandem repeat region. Similar to other haptophytes and rhodophytes, but not cryptophytes or stramenopiles, the mitochondrial genome has lost the nad7, nad9 and nad11 genes. The ~105 kb chloroplast genome encodes 112 protein coding genes, including ycf39 which has strong structural homology to NADP-binding nitrate transcriptional regulators; a divergent 'CheY-like' two-component response regulator (ycf55) and Tic/Toc (ycf60 and ycf80) membrane transporters. Notably, a zinc finger domain has been identified in the rpl36 ribosomal protein gene of all chloroplasts sequenced to date with the exception of haptophytes and cryptophytes--algae that have gained (via lateral gene transfer) an alternative rpl36 lacking the zinc finger motif. The two C. tobin chloroplast ribosomal RNA operon spacer regions differ in tRNA content. Additionally, each ribosomal operon contains multiple single nucleotide polymorphisms (SNPs)--a pattern observed in rhodophytes and cryptophytes, but few stramenopiles. Analysis of small (<200 bp) chloroplast encoded tandem and inverted repeats in C. tobin and 78 other algal chloroplast genomes show that repeat type, size and location are correlated with gene identity and taxonomic clade. CONCLUSION: The Chrysochromulina tobin organellar genomes provide new insight into organellar function and evolution. These are the first organellar genomes to be determined for the prymnesiales, a taxon that is present in both oceanic and freshwater systems and represents major primary photosynthetic producers and contributors to global ecosystem stability.

Targeted, haplotype-resolved resequencing of long segments of the human genome.

Currently, challenges exist to acquire long-range (hundreds of kilobase pairs) phase-discriminated sequence across substantial numbers of individuals. We have developed a straightforward method for isolating and characterizing specific genomic regions in a haplospecific manner. Real-time PCR is carried out to STS content map and genotype pools of fosmid clones arrayed in 384-well microtiter plates. Single-nucleotide polymorphisms, microsatellite markers, and insertion-deletion polymorphisms are used to differentiate the target region into haplotype-specific tiling paths. DNA of clones from these tiling paths is retrieved from the library and either sequenced by standard shotgun methods or amplified in vitro and sequenced by a primer-based, directed method. This approach provides convenient access to complete, haplotype-resolved resequencing data from multiple individuals across tens to hundreds of thousands of basepairs. We illustrate its implementation with a detailed example of more than 400 kbp from the human CFTR region, across 15 individuals, and summarize our experience applying it to many other human loci.