Labeled Peptide Nucleic Acid Research Articles

Reactive Quantum Dot-Based FRET Systems for Target-Catalyzed Detection of RNA.

Oligonucleotide-templated reactions (OTRs) between two reactive hybridization probes allow for the detection of a DNA or RNA of interest by exploiting the target molecule as a catalyst of chemical reactions. The product of such a reaction commonly exhibits distinct fluorescence properties and can be detected by the means of fluorescence spectroscopy. The vast majority of OTR systems utilize organic dyes as fluorescent reporters. However, the use of brighter emitters, such as semiconductor quantum dots (QDs), has potential to improve the sensitivity of detection by providing brighter signals and permitting the use of probes at very low concentrations. Here we report an RNA-templated reaction between two fluorescently labeled peptide nucleic acid (PNA)-based probes, which proceeds on the surface of a QD. The QD-bound PNA probe bears a cysteine functionality, while the other PNA is functionalized with an organic dye as a thioester. OTR between these probes proceeds through a transfer of the organic dye to the QD and can be conveniently monitored via fluorescence resonance energy transfer (FRET) from the QD to the Cy5. The reaction was performed in a conventional fluorescence microplate reader and permits the detection of RNA in the picomolar range.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans.

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)(1). Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT(2). Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Fluorometric Detection of MicroRNA Using Isothermal Gene Amplification and Graphene Oxide

We have developed a facile fluorometric system for the detection of microRNA (miRNA), using rolling circle amplification (RCA), graphene oxide (GO), and fluorescently labeled peptide nucleic acid (F-PNA). The padlock probe DNA complementary to a target miRNA was selectively ligated to form circular DNA that was then used as the template for RCA. F-PNAs complementary to the target miRNA were annealed to multiple sites of the isothermally amplified single-stranded RCA product (RCAP) containing multiple target miRNA sequences. This F-PNA/RCAP duplex is less adsorbed onto the GO monolayer, thus attenuating the quenching of F-PNA fluorescence by GO. In the absence of target miRNA (and hence the absence of RCA and duplex formation), the free F-PNA is completely adsorbed onto the GO monolayer and fluorescence quenching ensues. Thus, GO-based fluorescence detection coupled with isothermal gene amplification would be a simple and convenient method for the quantitative detection of miRNA.

Facile DNA Detection Based on Fluorescence Switching of A Hydrophobic AIE Dye-Labeled Peptide Nucleic Acid Probe by Aggregation/Disaggregation

A novel, facile DNA detection method based on aggregation/ disaggregation of an AIE (aggregation induced emission) dye- labeled peptide nucleic acid (PNA) probe was developed. This PNA probe plays the role of both target recognition and signal emission, thus it can quantify the target DNA without requiring additives such as intercalators and can distinguish the target from other sequences.

Arctic Circle Antibiotic Stewardship: An intervention with rapidly reduced in-patient antibiotic use

Background: Candida species are the fourth most common bloodstream infection with the highest crude mortality rate. Rapid identificationof thesepathogensdirectly fromnewlypositiveblood cultures (BC) can impact patient management by providing early appropriate empiric antifungal therapy. This study evaluated the impact of rapid detection of the predominant causes of candidemia, namely Candida albicans (CA) and Candida glabrata (CG), using the C. albicans/C. glabrata PNA FISH assay (AdvanDx Inc.;Woburn, MA). The effect of pathogen detection in real time on the selection of or change to appropriate antifungal treatment was measured. Real time differentiation of fluconazole susceptible CA strains from fluconazole non-susceptible CG is an important component of an effective antibiotic stewardship programand can beused as a guide to empiric therapy. Methods &Materials: Fluorescent labeled Peptide Nucleic Acid (PNA) probes targeting specific 26S rRNA sequences of CA and CG are utilized in the fluorescence in situ hydridization dual color C. albicans/C. glabrata PNA FISH assay. CA and CG are identified as brightgreenor redfluorescent cells, respectively. Identificationwas compared to conventional culture methods. Patient records were reviewed retrospectively for therapeutic changes based on rapid reporting. Results: FromJan throughNov 2013, 72 newly positive BCswith Gram stains positive for yeast, collected from 42 patients, were tested. The PNA FISH accurately identified 32 patients with BCs containing CA and/or CG, representing 76% of fungal BSIs, within 90minutes compared to 24-48hours by culture. Of the remaining 10 patients, the majority were infected with Candida parapsilosis (7). The sensitivity, specificity, positive and negative predictive values for the assay compared to culture were 100%. Rapid reporting resulted in therapy modifications for 42% of patients positive for CA and 71% of patients positive for CG. Conclusion: C. albicans/C. glabrata PNA FISH is a highly sensitive and specific assay for identifying CA and CG from BCs in real-time (90minutes compared to 1-3 days with culture). The ability to rapidly obtain species specific results is clinically useful in targeting appropriate pathogen specific therapy earlier in the course of candidemia

Dual fluorophore PNA FIT-probes − extremely responsive and bright hybridization probes for the sensitive detection of DNA and RNA

Fluorescently labeled oligonucleotides are commonly employed as probes to detect specific DNA or RNA sequences in homogeneous solution. Useful probes should experience strong increases in fluorescent emission upon hybridization with the target. We developed dual labeled peptide nucleic acid probes, which signal the presence of complementary DNA or RNA by up to 450-fold enhancements of fluorescence intensity. This enabled the very sensitive detection of a DNA target (40 pM LOD), which was detectable at less than 0.1% of the beacon concentration. In contrast to existing DNA-based molecular beacons, this PNA-based method does not require a stem sequence to enforce dye-dye communication. Rather, the method relies on the energy transfer between a "smart" thiazole orange (TO) nucleotide, which requires formation of the probe-target complex in order to become fluorescent, and terminally appended acceptor dyes. To improve upon fluorescence responsiveness the energy pathways were dissected. Hydrophobic, spectrally mismatched dye combinations allowed significant (99.97%) decreases of background emission in the absence of a target. By contrast, spectral overlap between TO donor emission and acceptor excitation enabled extremely bright FRET signals. This and the large apparent Stokes shift (82 nm) suggests potential applications in the detection of specific RNA targets in biogenic matrices without the need of sample pre-processing prior to detection.

Analysis of repetitive DNA in chromosomes by flow cytometry

We developed a flow cytometry method, chromosome flow fluorescence in situ hybridization (FISH), called CFF, to analyze repetitive DNA in chromosomes using FISH with directly labeled peptide nucleic acid (PNA) probes. We used CFF to measure the abundance of interstitial telomeric sequences in Chinese hamster chromosomes and major satellite sequences in mouse chromosomes. Using CFF we also identified parental homologs of human chromosome 18 with different amounts of repetitive DNA.

Metal-Enhanced Fluorescence of Conjugated Polyelectrolytes with Self-Assembled Silver Nanoparticle Platforms

We report the use of a simple Ag nanoparticle (NP) platform to enhance the fluorescence signatures of conjugated polyelectrolytes (CPEs). Ag NP platforms with different extinction intensities were fabricated by self-assembly of Ag NPs on NH(3)(+)-functionalized glass surface. Layer-by-layer (LBL) deposition of oppositely charged polyelectrolytes is used to control the Ag NP-CPE distance. The Ag NP platforms with high optical densities provide higher fluorescence enhancement factors for CPEs as compared to those with low optical densities. In addition, the CPE fluorescence enhancement is found to be directly related to the overlap between the absorption spectra of CPEs and the extinction spectra of Ag NP platforms. Both steady-state and time-resolved fluorescence spectroscopic studies reveal that the fluorescence enhancement is controlled by the increase in both absorption and radiative decay rates of the CPEs in proximity of Ag NPs. The enhanced CPE fluorescence signature is further used to detect single-stranded DNA using a Cy5 dye labeled peptide nucleic acid probe (Cy5-PNA) through Förster resonance energy transfer (FRET). We anticipate that the organic-inorganic hybrid platform will provide new opportunities for CPE application in sensing and device fabrication.

Localization of αII Spectrin to Telomeres After DNA Interstrand Cross-Link Damage Is Deficient In Fanconi Anemia Group A Cells, Which May Lead to Telomere Dysfunction.

AbstractAbstract 1160A hallmark of the bone marrow failure disorder, Fanconi anemia (FA), is genomic instability which is characterized by both chromosome instability and defects in repair of DNA interstrand cross-links (ICLs). The chromosome instability in FA, in particular chromosome end fusions and breaks, suggests that there is telomere dysfunction. We have shown that the structural protein, nonerythroid a spectrin (aIISp), is present in the nucleus of normal human cells where it is critical in both repair of DNA ICLS and maintaining chromosome stability. aIISp is important in recruitment of the ICL repair protein, XPF, to sites of damage. We have shown that there is a deficiency in aIISp in FA cells lines, due to its increased breakdown, which correlates with reduced repair of DNA ICLs and increased chromosomal aberrations after ICL damage. The goal of the present study was to determine whether aIISp plays a role in telomere function after DNA ICL damage and whether telomere dysfunction in FA cells after damage can be corrected when levels of aIISp are restored to normal. Normal human and FA, complementation group A (FA-A) lymphoblastoid cells, which have telomerase, were used. Undamaged cells or cells treated with mitomycin C (MMC), a DNA ICL agent, were studied using immunofluorescence and in situ hybridization (IF-FISH). Cells were probed with anti-a-spectrin and either (C3TA2)3-Cy3 labeled peptide nucleic acid (PNA) telomeric probe or antibodies against two proteins, TRF1 or TRF2, which are components of the shelterin complex that is essential for protecting chromosome ends and preventing telomere dysfunction. The appropriate fluorescent secondary antibodies were used. The results showed that 47–50% of aIISp foci colocalized with the PNA probe, TRF1 or TRF2 in telomeres in MMC damaged but not undamaged normal cells. Levels of colocalization peaked at 16 hours and, by 24 hours, aIISp foci were no longer observed. This colocalization was specific for the S phase of the cell cycle. These results showed that in normal cells aIISp associates with telomeres in S phase after ICL damage and may play an important role in telomere function after damage. After ICLs are presumably unhooked/removed, aIISp dissociates from telomeres. The view that aIISp is involved in telomere function after ICL damage is strengthened by coimmunoprecipitation studies which show that aIISp has markedly enhanced binding for TRF1 and TFR2 after cells were damaged with MMC. IF-FISH also showed that the ICL repair protein, XPF, had enhanced association with telomeres after ICL damage, which further indicated that repair is taking place. In FA-A cells, in which aIISp levels are reduced, aIISp did not colocalize with the PNA probe, with TRF1 or with TRF2 after ICL damage; XPF also did not localize to telomeres after ICL formation. However, after aIISp levels were returned to normal in FA-A cells by siRNA knock down of m-calpain, a protease which cleaves aIISp and levels of which are elevated in FA cells, aIISp as well as XPF associated with telomeres after ICL damage. The chromosomal aberrations (i.e., breaks and end fusions) observed in FA-A cells after ICL damage were also corrected. ICLs can lead to stalled DNA replication forks in S phase. The present results suggest that, after damage, aIISp associates with telomeres in S phase at ICLs at stalled replication forks. Since aIISp is involved in ICL repair, this association could be of particular importance in repair of ICLs in replicating telomeres. In FA-A cells, failure of localization of aIISp and XPF to telomeres after ICL damage could have severe consequences. It could lead to unrepaired ICLs, which in replicating telomeres could lead to telomere dysfunction and to production of the chromosomal aberrations observed in these cells. These studies show that aIISp may play an important role in telomere function after DNA damage and during telomere replication. Correction of the deficiency in aIISp in FA cells by knocking down m-calpain could be significant in reversing telomere dysfunction and chromosomal aberrations after ICL damage and could potentially have important therapeutic relevance. Disclosures:No relevant conflicts of interest to declare.

VEGFA amplification/deletion in human breast tumors.

e21017 Background: The anti-VEGF antibody, bevacizumab, has been FDA approved for the treatment of breast cancer. While germline variability (i.e. single nucleotide polymorphisms) may serve as a predictive marker for anti-VEGF therapy, to date tumor-specific variability has not. More specifically, amplification or deletion of the VEGFA gene has not been studied or evaluated as a predictive or prognostic biomarker for breast cancer. Methods: A VEGFA/centromere enumerization-6 (CEN-6) probe was created and validated using DNA clones and restriction enzyme fragment measurements. The final product contained a RP11-710-L16 probe covering 183 KB including the VEGFA gene and flanking regions. The CEN-6 probe was developed with fluorescein isothiocyanate labeled peptide nucleic acid oligonucleotides. VEGFA and CEN-6 probes were tested on metaphase spreads to exclude cross-hybridization to other chromosomes. A tissue microarray containing 93 breast cancers was analyzed for the presence of VEGFA gene amplification and/or deletion using a FISH protocol similar to the TOP2A FISH pharmDx Kit. VEGFA/CEN-6 signal ratio in cancer cells was recorded. Normal cells in the tissue sections served as an internal positive control of pretreatment and hybridization efficiency. A ratio of <0.8 was considered deleted, ≥ 1.5-1.99 was considered borderline amplified, whereas a ratio≥ 2.0 was considered amplified. Results: Of the 93 core tissue specimens, FISH analysis was successful in 80 cases (86%) of the cases. Of these, 11% were found to demonstrate VEGFA deletions, 9% were borderline amplified and 5% were amplified. Thus, 25% in total had genetic aberrations of this gene. The aberrations did not correlate with ER or HER2 expression, tumor size, nodal status or grade. Conclusions: VEGFA gene aberrations are relatively common in primary breast cancers. Their role as predictors of response to anti-VEGF therapies remains to be studied. Result n = 80 % Deletion (<0.8) 9 11 Normal (0.8-1.49) 60 75 Borderline (1.5-1.99) 7 9 Amplified (≥2.0) 4 5 Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Dako Denmark A/S Molecular Pathology

Real Time Electrochemical Monitoring of DNA/PNA Dissociation by Melting Curve Analysis

AbstractAn immobilization‐free electrochemical method is reported for real‐time monitoring of the DNA hybrid dissociation between a ferrocene labeled peptide nucleic acid (PNA) and a fully‐complementary or single‐base‐mismatched DNA. This method takes advantages of electrostatic charge characteristics and interactions among the neutrally charged PNA, the negatively charged DNA and the negatively charged electrode surface made of indium tin oxide (ITO). When a ferrocene labeled PNA (Fc‐PNA) sequence is hybridized to a complementary DNA strand, electrostatic repulsion between the negatively charged PNA/DNA hybrid and the negative ITO surface retards the diffusion of the electroactive Fc to the electrode, resulting in a much reduced electrochemical signal. On the other hand, when the Fc‐PNA is dissociated from the hybrid at elevated temperatures, the neutrally charged Fc‐PNA easily diffuses to the electrode with an enhanced electrochemical signal. Therefore, an electrochemical melting curve of the Fc‐PNA/DNA hybrid can be obtained by measuring the Fc signal with the increasing temperature. This strategy allows monitoring of the dissociation of the DNA hybrid in real time, which might lead to a simple detection method for single nucleotide polymorphism (SNP) analysis.

A FRET-based assay for characterization of alternative splicing events using peptide nucleic acid fluorescence in situ hybridization

We describe a quantitative method for detecting RNA alternative splicing variants that combines in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes with confocal microscopy Förster resonance energy transfer (FRET). The use of PNA probes complementary to sequences flanking a given splice junction allows to specifically quantify, within the cell, the RNA isoform generating such splice junction by FRET measure. As a proof of concept we analyzed two alternative splicing events originating from lymphocyte antigen 6 (LY6) complex, locus G5B (LY6G5B) pre-mRNA. These are characterized by the removal of the first intron (Fully Spliced Isoform, FSI) or by retention of such intron (Intron-Retained Isoform, IRI). The use of PNA probe pairs labeled with donor (Cy3) and acceptor (Cy5) fluorophores, suitable to FRET, flanking FSI and IRI specific splice junctions specifically detected both mRNA isoforms in HeLa cells. We have observed that the method works efficiently with probes 5–11 nt apart. The data supports that this FRET-based PNA fluorescence in situ hybridization (FP–FISH) method offers a conceptually new approach for characterizing at the subcellular level not only splice variant isoform structure, location and dynamics but also potentially a wide variety of close range RNA–RNA interactions.

Property and application of novel ferrocenyl–azobenzene labeled peptide nucleic acid monomers with the dual stimulus–response characteristics

In this work, three novel types of peptide nucleic acid (PNA) monomers labeled by ferrocenyl–azobenzene units (Fc–Azo–PNAs, 1a, 1b, and 1c) have been synthesized and they all exhibited good reversible photoswitch and redox properties. In the different solvents of ethanol (EtOH) and acetonitrile (ACN), photoisomerization rate constants ( K) of three PNA monomers under UV light irradiation have also been analyzed and compared, discovering that the values of K are closely correlative to their molecular structures. The electrochemical properties of the synthesized PNA monomers are also investigated and the typical redox properties are displayed, demonstrating that they can be regarded as the anchor to be grafted at the end of PNA oligomers for detecting themselves. Finally, according to the analyses of UV–Vis absorption spectrum and differential pulse voltammetry (DPV), the synthesized PNA monomers afford the best detection limit down to 10 −8 M in EtOH. The results also illuminate that PNA monomers with new photo–redox dual stimulus–response characteristics can be realized to detect themselves by the metal complex unit together with azo moiety; moreover, the hybridization with DNA also can be tuned by the isomerization of azo moiety.

Flow cytometry and FISH to measure the average length of telomeres (flow FISH)

Telomeres have emerged as crucial cellular elements in aging and various diseases including cancer. To measure the average length of telomere repeats in cells, we describe our protocols that use fluorescent in situ hybridization (FISH) with labeled peptide nucleic acid (PNA) probes specific for telomere repeats in combination with fluorescence measurements by flow cytometry (flow FISH). Flow FISH analysis can be performed using commercially available flow cytometers, and has the unique advantage over other methods for measuring telomere length of providing multi-parameter information on the length of telomere repeats in thousands of individual cells. The accuracy and reproducibility of the measurements is augmented by the automation of most pipetting (aspiration and dispensing) steps, and by including an internal standard (control cells) with a known telomere length in every tube. The basic protocol for the analysis of nucleated blood cells from 22 different individuals takes about 12 h spread over 2-3 days.

New synthesis of a spin-labeled peptide nucleic acid and its interactions with nucleic acids

A new combined solid-liquid phase synthesis method for a spin labeled peptide nucleic acid (PNA) is developed. The methodology involved initial preparation of a protected PNA on solid phase, followed by efficient solution phase coupling to a spin label containing a reactive carboxylic group. This strategy allows to maintain the integrity of the nitroxide moiety during the various steps of chemical synthesis assuring in the same time the fidelity of the hybridization assay. This compound can be used as a reporter molecule to investigate the binding of peptide nucleic acids to oligonucleotide sequences (DNA or RNA) by EPR spectroscopy.

GROWTH REGULATION OF rRNA CONTENT IN PROCHLOROCOCCUS AND SYNECHOCOCCUS (MARINE CYANOBACTERIA) MEASURED BY WHOLE‐CELL HYBRIDIZATION OF rRNA‐TARGETED PEPTIDE NUCLEIC ACIDS1

The cyanobacteria Synechococcus and Prochlorococcus are important primary producers in marine ecosystems. Because currently available approaches for estimating microbial growth rates can be difficult to apply in the field, we have been exploring the feasibility of using quantitative rRNA measurements as the basis for making such estimates. In this study we examined the relationship between rRNA and growth rate in several Synechococcus and Prochlorococcus strains over a range of light‐regulated growth rates. Whole‐cell hybridization with fluorescently labeled peptide nucleic acid (PNA) probes was used in conjunction with flow cytometry to quantify rRNA on a per cell basis. This PNA probing technique allowed rRNA analysis in a phycoerythrin‐containing Synechococcus strain (WH7803) and in a non–phycoerythrin‐containing strain and in Prochlorococcus. All the strains showed a qualitatively similar tri‐phasic relationship between rRNA·cell−1 and growth rate, involving relatively little change in rRNA·cell−1 at low growth rates, linear increase at intermediate growth rates, and a plateau and/or decrease at the highest growth rates. The onset of each phase was associated with the relative, rather than absolute, growth rate of each strain. In the Synechococcus strains, rRNA normalized to flow cytometrically measured forward angle light scatter (an indicator of size) was well‐correlated with growth rate across strains. These findings support the idea that cellular rRNA may be useful as an indicator of in situ growth rate in natural Synechococcus and Prochlorococcus populations.

Synthesis of chiral chromium tricarbonyl labeled thymine PNA monomers via the Ugi reaction.

[reaction: see text] Ugi condensation was used to synthesize the first examples of chiral racemic Ar.Cr(CO)(3) labeled peptide nucleic acid (PNA) monomers bearing the organometallic moiety linked to the alpha-carbon of the glycine unit.

Simultaneous flow cytometric analysis of two cell surface markers, telomere length, and DNA content.

Various protocols for estimation of telomere length in individual cells by flow cytometry using fluorescence in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes (Flow-FISH) have been described. Combined analysis of telomere length and cell phenotype, however, remains difficult because few fluorochromes with suitable emission spectra tolerate the harsh conditions needed for DNA denaturation during hybridization of the telomere-specific PNA probe. We overcame these problems and developed a method for measuring telomere length in cell subsets characterized by the expression of two surface antigens. Alexa Fluor 488 and Alexa Fluor 546 were used for cell surface staining. Antigen-antibody complexes were covalently cross-linked onto the cell membrane before Flow-FISH. Cells were hybridized with a PNA probe conjugated to cyanine 5 (Cy5). Hoechst 33342 (HO342) was added for determination of cellular DNA content. For assay standardization, we added an aliquot of a single batch of 1,301 cells to each sample as an internal control before hybridization with the PNA probe. Samples were prepared in duplicate and analyzed on a standard three-laser BD LSR flow cytometer. For assay validation, the same samples were analyzed in parallel to correlate the percentage of telomere length of the sample versus 1,301 control cells to the mean size of terminal restriction fragments (TRFs) of DNA as determined by Southern gel analysis. The method permitted clear identification of lymphocyte subsets in samples hybridized for Flow-FISH, with subset frequencies comparable to those of untreated samples. At a concentration of 10 nM, the Cy5-labeled telomere-specific PNA probe produced a bright fluorescence signal well separated from background. Addition of HO342 in low concentration did not interfere with Cy5 telomere fluorescence, produced adequate DNA histograms, and permitted clear identification of cell phenotype. The probe concentration of 10 nM also proved optimal for inclusion of 1,301 control cells for assay standardization. Telomere length estimations by the current method correlated highly with TRF calculations by Southern gel hybridization (r(2)= 0.9, P = 0.0003). Application of our protocol to the analysis of human CD8CD28 lymphocyte subsets showed that CD8(+bright)CD28(-) lymphocytes generally exhibit shorter telomeres than CD8(+bright)CD28(+) cells. These data concurred with previous results of telomere shortening in CD8(+)CD28(-) T cells that were obtained by using different techniques. The multiparameter Flow-FISH protocol permitted rapid determination of differences in telomere length in subpopulations characterized by two surface markers without prior cell separation.

LIFE CYCLE OF THE HETEROTROPHIC DINOFLAGELLATE PFIESTERIA PISCICIDA (DINOPHYCEAE)1

The putatively toxic dinoflagellate Pfiesteria piscicida (Steidinger et Burkholder) has been reported to have an unusual life cycle for a free-living marine dinoflagellate. As many as 24 life cycle stages were originally described for this species. During a recent phylogenetic study in which we used clonal cultures of P. piscicida, we were unable to confirm many reported life cycle stages. To resolve this discrepancy, we undertook a rigorous examination of the life cycle of P. piscicida using nuclear staining techniques combined with traditional light microscopy, high-resolution video microscopy, EM, and in situ hybridization with a suite of fluorescently labeled peptide nucleic acid (PNA) probes. The results showed that P. piscicida had a typical haplontic dinoflagellate life cycle. Asexual division occurred within a division cyst and not by binary fission of motile cells. Sexual reproduction of this homothallic species occurred via the fusion of isogamous gametes. Examination of tanks where P. piscicida was actively feeding on fish showed that amoebae were present; however, they were contaminants introduced with the fish. Whole cell probing using in situ hybridization techniques confirmed that these amoebae were hybridization negative for a P. piscicida-specific PNA probe. Direct observations of clonal P. piscicida cultures revealed no unusual life cycle stages. Furthermore, the results of this study provided no evidence for transformations to amoebae. We therefore conclude that P. piscicida has a life cycle typical of free-living marine dinoflagellates and lacks any amoeboid or other specious stages.

Measurements of telomere length on individual chromosomes by image cytometry

Publisher Summary The physical ends of chromosomes or telomeres are in most species composed of G-rich repeat sequences and associated proteins. The DNA of telomeres consists of specific repetitive sequences (TTAGGG in all vertebrates) that are synthesized from an RNA template by a specialized reverse transcriptase called telomerase. Conventionally, Southern analysis is used to estimate the average length of telomere repeat sequences. For this purpose, DNA extracted from a population of cells is digested with restriction enzymes and analyzed by gel electrophoresis. With this method DNA fragments are separated on the basis of size. The telomere repeat sequences present in a minor fraction of the DNA fragments are visualized after hybridization with a radiolabeled probe specific for telomeric DNA. Information on the length of telomere repeats in individual chromosomes can also be extracted from digital images of metaphase chromosomes that are subjected to quantitative fluorescence in situ hybridization (Q-FISH) with directly labeled peptide nucleic acid (PNA) telomere probes.