Karenia brevis is a toxic marine dinoflagellate endemic to the Gulf of Mexico. Blooms of this harmful alga cause fish kills, marine mammal mortalities and neurotoxic shellfish poisonings. These harmful effects are attributed to a suite of polyketide secondary metabolites known as the brevetoxins. The carbon framework of all polyketides is assembled by a polyketide synthase (PKS). Previously, PKS encoding genes were amplified from K. brevis culture and their similarity to a PKS gene from the closely related protist, Cryptosporidium parvum, suggested that these genes originate from the dinoflagellate. However, K. brevis has not been grown axenically. The associated bacteria might be the source of the toxins or the PKS genes. Herein we report the localization of PKS encoding genes by a combination of flow cytometry/PCR and fluorescence in situ hybridization (FISH). Two genes localized exclusively to K. brevis cells while a third localized to both K. brevis and associated bacteria. While these genes have not yet been linked to toxin production, the work describes the first definitive evidence of resident PKS genes in any dinoflagellate.

Electronic cell sorting for isolation and culture of dinoflagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking cells using a micropipette. Trauma to electronically sorted cells was not a limiting factor, as fragile dinoflagellates, such as Karenia brevis (Dinophyceae), survived electronic cell sorting to yield viable cells. The rate of successful isolation of large-scale (> 4 litres) cultures was higher for manual picking than for electronic cell sorting (2% vs 0.5%, respectively). However, manual picking of cells is more labor intensive and time consuming. Most manually isolated cells required repicking, as the cultures were determined not to be unialgal after a single round of isolation; whereas, no cultures obtained in this study from electronic single-cell sorting required resorting. A broad flow cytometric gating logic was employed to enhance species diversity. The percentages of unique genotypes produced by manual picking or electronic cell sorting were similar (57% vs 54%, respectively), and each approach produced a variety of dinoflagellate or raphidophyte genera. Alternatively, a highly restrictive gating logic was successfully used to target K. brevis from a natural bloom sample. Direct electronic single-cell sorting was more successful than utilizing a pre-enrichment sort followed by electronic single-cell sorting. The appropriate recovery medium may enhance the rate of successful isolations. Seventy percent of isolated cells were recovered in a new medium (RE) reported here, which was optimized for axenic dinoflagellate cultures. The greatest limiting factor to the throughput of electronic cell sorting is the need for manual postsort culture maintenance and assessment of the large number of isolated cells. However, when combined with newly developed automated methods for growth screening, electronic single-cell sorting has the potential to accelerate the discovery of new algal strains.

Extracts of fifty-seven newly isolated strains of dinoflagellates and raphidophytes were screened for protein phosphatase (PP2A) inhibition. Five strains, identified by rDNA sequence analysis as Prorocentrum rhathymum, tested positive and the presence of okadaic acid was confirmed in one strain by HPLC-MS/MS and by HPLC with fluorescence detection and HPLC-MS of the okadaic acid ADAM derivative. Quantitation of the ADAM derivative indicated that the concentration of okadaic acid in the culture medium is 0.153 μg/L. KeywordsProrocentrum rhathymum; okadaic acid; diarrheic shellfish poisoning (DSP) toxins; LC-MS; ADAM derivative; HPLC-FLD The suite of marine toxins that includes okadaic (OA) acid and the dinophysistoxins (DTX) is collectively known as DSP (diarrheic shellfish poisoning) toxins. The acute symptoms of DSP include diarrhea, nausea, vomiting and abdominal pain. Outbreaks have been documented worldwide and are associated with the consumption of mussels, scallops, or clams tainted with OA, its analogs or derivatives. (Gestal-Otero, 2000). OA has been isolated from several dinoflagellates in the genera Prorocentrum and Dinophysis, including the species P. lima (Yasumoto et al., 1987), P. hoffmanianum (Murakami et al., 1982), P. concavum (Dickey et al., 1990), P. maculosum (Zhou and Fritz, 1993), P. belizeanum , P. faustiae (Morton 1998), P. arenarium (Ten-Hage et al., 2000), D. acuta (Draisci et al., 1998) and D. fortii (Murata et al., 1982). In addition to OA, several related polyethers were isolated from these dinoflagellates, including the dinophysis toxins, DTX-1 and DTX-2 which differ in the location and number of methyl substituents (Murata et al., 1982;Yasumoto, et al., 1985;Hu et al., 1993). OA, DTX-1 and DTX-2 are inhibitors of protein phosphatases PP1 and PP2A (Dounay and Forsyth, 2002 We recently reported the isolation of over fifty strains of dinoflagellates by viable high speed single-cell sorting ). Five of these strains (6-9 and 25) matched most closely with P. rhathymum in a BLAST (Altschul et al., 1990) analysis of their large-subunit ribosomal genes ) and tested positive for protein phosphatase (PP2A) inhibition in preliminary screening. The presence of OA was confirmed in one strain by HPLC with fluorescent detection of the ADAM (9-anthryldiazomethane) derivative (Quilliam et al., 1998) and by HPLC-MS and MS/MS experiments.Fifty-seven field strains were sequenced in the D1/D2 region of the LSU rDNA and compared with GenBank sequence data ). Dinoflagellate and raphidophyte genera identified included Akashiwo, Amphidinium, Heterocapsa, Cachonina, Chatonella, Coolia, Fragilidinium, Heterosigma, Karlodinium, Karenia, Protoceratium, Scripsciella and Prorocentrum. Field strains 6, 7, 8, 9, 23, 25 and identified strains CCMP687 (Prorocentrum mexicanum) and CCMP1591 (Prorocentrum micans) clustered within a clade of the genus Prorocentrum (Fig. 1). The sequences for strains 6, 7, 8 and 9 were identical to each other and matched with two sequences (AY259166 and AY259167) of P. rhathymu...

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DNA displacement synthesis by reverse transcriptase during retroviral replication is required for the production of the linear precursor to integration. The sensitivity of unpaired thymines to KMnO 4 oxidation was used to probe for the extent of DNA melting by human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase in front of the primer terminus in model oligonucleotide-based displacement constructs. Unpairing of the two base pairs downstream of the primer (؉1 and ؉2 positions) requires the presence of the next correct dNTP, indicating that DNA melting only occurs after the formation of the ternary complex with the enzyme tightly clamped around the DNA. The amount or extent of DNA melting is not significantly affected by the length of the already-displaced strand or the base composition of the DNA beyond the ؉2 position. The F61W mutant form of HIV-1 reverse transcriptase, which is partially impaired for displacement synthesis, exhibits a reduction in the amount of melting at the ؉1 and ؉2 positions. These results demonstrate the importance of the observed melting to displacement synthesis and suggest that the unpairing reaction is mediated by an intimate association between the fingers region of the enzyme and the DNA in the closed clamp conformation of the protein. HIV-11 reverse transcriptase is a multifunctional enzyme with a polymerase domain that catalyzes both RNA-dependent and DNA-dependent DNA synthesis and an RNase H domain that catalyzes degradation of RNA when it is hybridized to DNA (for reviews, see Refs. 1 and 2). Viral reverse transcription is initiated from a specific tRNA primer that anneals near the 5Ј-end of the plus-sense RNA genome at the primer binding site. Extension of the tRNA primer followed by the first strand transfer allows the completion of the minus DNA strand and generates the substrate for the RNase H cleavage that creates the plus-strand primer at the polypurine tract. Removal of the tRNA allows a second strand transfer that occurs via complementary primer binding site regions at the 3Ј-ends of both plus and minus strands. Completion of both plus and minus strands to produce the long terminal repeat-flanked linear product required for integration is believed to occur through a circular intermediate in which reverse transcriptase must carry out strand displacement synthesis through a stretch of DNA ϳ600 bp in length (3, 4).Reverse transcriptases have been shown to be capable of displacing either a DNA or an RNA non-template strand ahead of the primer terminus during polymerase chain elongation in vitro (4 -10), and there is evidence for displacement synthesis during plus-strand synthesis in permeabilized virions (11-13). In addition, RNA displacement synthesis may be required for removal of stably annealed fragments of the RNA genome after minus strand DNA synthesis (14).HIV-1 reverse transcriptase is a heterodimer, consisting of 66-and 51-kDa subunits that share the same N terminus. Proteolytic cleavage of the p66 subunit, which removes the C-terminal RNase H domain...