John J. Gaynor

Molecular cloning, characterization and expression of Mn-superoxide dismutase from the rubber tree (Hevea brasiliensis)

A genomic clone encoding manganese-containing superoxide dismutase (SOD; EC 1.15.1.1) was isolated from a Hevea brasiliensis genomic library made in λ phage EMBL3 by using a heterologous cDNA probe of MnSOD from Nicotiana plumbaginifolia. The nucleotide sequence of 4968 bp from the genomic clone was determined. Based on the putative translation initiation codon and stop codon, PCR primers were designed and utilized for cloning the full-length cDNA from total mRNA. Of the two distinct cDNAs of MnSOD isolated, MnSOD-A has a perfect match with exons of the nuclear gene, while MnSOD-B has a 90.2% homology and is 6 nucleotides longer than MnSOD-A in the putative transit peptide region. The nuclear gene comprises 6 exons and 5 introns, giving a total length of 3211 bp. The sequences of 1400 bp upstream of the initiation codon and 320 bp downstream of the stop codon were also determined. Southern analysis of genomic DNA from Hevea probed with a genomic fragment indicated there are at least two genes of MnSOD in Hevea. Northern blot analysis showed that MnSOD transcripts were present in all tissues examined (leaf, petiole, root, latex, callus) with young leaves showing the highest levels in intact plants. The transcript level in embryogenic callus was nearly 50-fold higher than in mature leaves. In addition, transcripts of MnSOD could be induced 3- to 5-fold in response to sucrose, ethephon and Murashige-Skoog salts.

Induction of temperate cyanophage AS-1 by heavy metal – copper

BackgroundIt has been reported that some marine cyanophage are temperate and can be induced from a lysogenic phase to a lytic phase by different agents such as heavy metals. However, to date no significant reports have focused on the temperate nature of freshwater cyanophage/cyanobacteria. Previous experiments with cyanophage AS-1 and cyanobacteria Anacystis nidulans have provided some evidence that AS-1 may have a lysogenic life cycle in addition to the characterized lytic cycle.ResultsIn this study, the possible temperate A. nidulans was treated with different concentrations of heavy metal-copper. CuSO4 with concentrations of 3.1 × 10-3 M, 3.1 × 10-4 M, 3.1 × 10-5 M and 3.1 × 10-6 M were used to detect the induction of AS-1 from A. nidulans. The population of the host, unicellular cyanobacteria Anacystis nidulans, was monitored by direct count and turbidity while the amount of virus produced was derived from plaque forming units (PFU) by a direct plating method. The ratio of AS-1 release from A. nidulans was also determined. From these results it appears that AS-1 lysogenic phage can be induced by copper at concentrations from 3.1 × 10-6 M to 3.1 × 10-4 M. Maximal phage induction occurred at 6 hours after addition of copper, with an optimal concentration of 3.1 × 10-6 M.ConclusionCu2+ is a significant inducer for lysogenic cyanobacterial cells and consequently would be a potential control agent in the cyanobacteria population in fresh water ecosystems.

Diet assessment of the Atlantic Sea Nettle Chrysaora quinquecirrha in Barnegat Bay, New Jersey, using next-generation sequencing

Next‐generation sequencing (NGS) methodologies have proven useful in deciphering the food items of generalist predators, but have yet to be applied to gelatinous animal gut and tentacle content. NGS can potentially supplement traditional methods of visual identification. Chrysaora quinquecirrha (Atlantic sea nettle) has progressively become more abundant in Mid‐Atlantic United States’ estuaries including Barnegat Bay (New Jersey), potentially having detrimental effects on both marine organisms and human enterprises. Full characterization of this predators diet is essential for a comprehensive understanding of its impact on the food web and its management. Here, we tested the efficacy of NGS for prey item determination in the Atlantic sea nettle. We implemented a NGS ‘shotgun’ approach to randomly sequence DNA fragments isolated from gut lavages and gastric pouch/tentacle picks of eight and 84 sea nettles, respectively. These results were verified by visual identification and co‐occurring plankton tows. Over 550 000 contigs were assembled from ~110 million paired‐end reads. Of these, 100 contigs were confidently assigned to 23 different taxa, including soft‐bodied organisms previously undocumented as prey species, including copepods, fish, ctenophores, anemones, amphipods, barnacles, shrimp, polychaete worms, flukes, flatworms, echinoderms, gastropods, bivalves and hemichordates. Our results not only indicate that a ‘shotgun’ NGS approach can supplement visual identification methods, but targeted enrichment of a specific amplicon/gene is not a prerequisite for identifying Atlantic sea nettle prey items.

A cDNA Encodes the Drosophila Homolog of Yeast 60S Ribosomal Protein YL43

We describe the nucleotide sequence of a cDNA clone isolated from Drosophila Kc cells which encodes an amino acid sequence homologous to a 60S ribosomal protein from yeast (YL43) and rat (p23). The DL43 cDNA is 320 nucleotides in length and predicts a protein of 76 amino acids and a calculated molecular mass of 8.9 kiloDaltons. Northern blot analysis demonstrates the presence of the DL43 transcript under both control (25 degrees C) and heat shock (37 degrees C) conditions. The Drosophila protein shares an 86% identity over the first 22 amino acids with the yeast YL43 protein and a 60% identity over the entire length of the partial sequence available for this protein.

Identification of a Drosophila 60S ribosomal protein.

We have previously reported the cloning and sequencing of a cDNA from Drosophila (DL43; GenBank Accession # U40226) which predicts a small protein homologous to a ribosomal protein in yeast and rat. In this study we show that a 330 nucleotide transcript encoding the putative ribosomal protein is expressed in Drosophila Kc cells under normal growth conditions (25 degrees C). This RNA is associated with the translational machinery and is present on polysomes. The DL43 cDNA was transcribed using T7 RNA polymerase and the resulting transcripts translated in a wheat germ extract. SDS/PAGE analysis of the in vitro transcribed and translated DL43 cDNA yields a small protein of approximately 9 kDa. Using two-dimensional gelelectrophoresis we have established that the radiolabeled DL43 protein comigrates with a small basic protein in the 60S ribosomal subunit. The electrophoretic mobilities of the in vitro and in vivo synthesized DL43 proteins are indistinguishable providing support for the identity of this protein.

Who’s lurking in your lagoon? First occurrence of the invasive hydrozoan Moerisia sp. (Cnidaria: Hydrozoa) in New Jersey, USA

Coastal estuaries represent areas of high biological invasions by virtue of their economic importance as ports. We report on the first occurrence of the non-native hydrozoan Moerisia sp. in coastal New Jersey, USA. Through the use of artificial settling plates, several diminutive, unknown cnidarian polyps were isolated. Initial morphological assessment indicated that two of the unknown polyps were keyed to Moerisia. We then used universal cnidarian primers to amplify and sequence the 16S rDNA mitochondrial locus for molecular identification. Upon evaluation and editing of sequences, two of the unknown polyps were identified as belonging to a group of unresolved Moerisia sp. taxa (> 99% homology). Additionally, polyps of Chrysaora and Aequorea were also identified from settling plates. The presence of Moerisia sp. in Barnegat Bay is the second recent documentation of an invasive hydrozoan in New Jersey and suggests that there may be other undescribed hydrozoans in this region that have yet to be been recognized, especially those with cryptic benthic life history phases.

qPCR Detection of Early Life History Stage Chrysaora quinquecirrha (Sea Nettles) in Barnegat Bay, New Jersey

ABSTRACT Gaynor, J.J.; Bologna, P.A.X.; Restaino, D.J., and Barry, C.L., 2017. qPCR detection of early life history stage Chrysaora quinquecirrha (sea nettles) in Barnegat Bay, New Jersey. In: Buchanan, G.A.; Belton, T.J., and Paudel, B. (eds.), A Comprehensive Assessment of Barnegat Bay–Little Egg Harbor, New Jersey. The sea nettle Chrysaora quinquecirrha has become abundant in the Barnegat Bay estuary and frequently blooms in warm summer months. Various factors have been attributed to the increasing localized appearance of sea nettles and other jellyfish including eutrophication, overfishing, global warming, construction, and species introduction. Despite its abundance and frequent distribution within estuarine systems, very little work has been done to detect and quantify the early life history stages of this organism. Free-swimming larval stages of C. quinquecirrha can be detected and quantified using a quantitative polymerase chain reaction assay specific for the C. quinquecirrha 16S ribosomal (r)DNA locus of the mitochondrial DNA. This assay is species specific, linear over a 9-log range, and can detect as few as 10 copies of 16S rDNA. Twenty-liter field samples were sequentially filtered through 500- and 100-μm mesh to separate ephyra from planula larvae and gametes, respectively. Quantifiable levels of C. quinquecirrha 16S rDNA were detected at all eight paired locations in Barnegat Bay, with levels varying on both spatial and temporal scales. This research is apparently the first comprehensive field-based survey mapping, both spatially and temporally, the early life history stages of a scyphozoan in a major estuary using environmental DNA. Quantitative molecular data on the distribution of early stage C. quinquecirrha may prove useful in both understanding and managing blooms of sea nettles in Barnegat Bay.

Top-Down Impacts of Sea Nettles (Chrysaora quinquecirrha) on Pelagic Community Structure in Barnegat Bay, New Jersey, U.S.A.

ABSTRACT Bologna, P.A.X.; Gaynor, J.J.; Barry C.L., and Restaino, D.J., 2017. Top-down impacts of sea nettles (Chrysaora quinquecirrha) on pelagic community structure in Barnegat Bay, New Jersey, U.S.A.. In: Buchanan, G.A.; Belton, T.J., and Paudel, B. (eds.), A Comprehensive Assessment of Barnegat Bay–Little Egg Harbor, New Jersey. Coastal communities are substantially affected by human activities and create environments conducive to opportunistic species and structural changes in food webs. The Mid-Atlantic coast of the United States is highly urbanized with significant landscape modification and elevated pollutant loads. The appearance and development of resident populations of the Atlantic sea nettle (Chrysaora quinquecirrha) in Barnegat Bay, New Jersey demonstrates a successful establishment to this estuary. This research indicates that two species of gelatinous zooplankton (Mnemiopsis leidyi, C. quinquecirrha) play important structuring roles in the pelagic community. Specifically, M. leidyi exerts significant top-down control of calanoid copepods, cladocerans, fish eggs, and fish larvae, whereas C. quinquecirrhas impact is felt through control of M. leidyi, whose density is two orders of magnitude greater. It was expected that if C. quinquecirrha exerted top-down control of M. leidyi, then a trophic cascade would result. However, no trophic cascade was observed, as C. quinquecirrha demonstrated broad control of pelagic community structure as a nonspecific, generalist predator. Consequently, the strength of M. leidyis ability to exert predation pressure is mediated by the development of the C. quinquecirrha bloom, but pelagic community structure is broadly defined by the combined impact of these predators within the system.

Uptake of bacteriophage λ DNA in Escherichia coli by electroporation : An alternative to in vitro packaging

By using a high field strength DC pulse of 15 kV/cm and a pulse duration of 5 ms for the transfection of E. coli by bacteriophage λ DNA, we obtained efficiencies of 1.1 × 106 (pfu/μg bacteriophage λ, DNA). This represents a 100-fold improvement over the traditional CaCl2/heat shock method and is a viable alternative to the more costly in vitro packaging of recombinant bacteriophage λ DNA for the production of cDNA and genomic libraries.