Showing papers in "Marine Biotechnology in 2004"
TL;DR: During 2000 pharmacologic research with marine chemicals continued to contribute potentially novel chemical leads to the ongoing global search for therapeutic agents in the treatment of multiple disease categories.
Abstract: During 2000 research on the pharmacology of marine chemicals involved investigators from Australia, Brazil, Canada, Egypt, France, Germany, India, Indonesia, Israel, Italy, Japan, the Netherlands, New Zealand, Phillipines, Singapore, Slovenia, South Korea, Spain, Sweden, Switzerland, United Kingdom, and the United States. This current review, a sequel to the authors’ 1998 and 1999 reviews, classifies 68 peer-reviewed articles on the basis of the reported preclinical pharmacologic properties of marine chemicals derived from a diverse group of marine animals, algae, fungi, and bacteria. Antibacterial, anticoagulant, antifungal, antimalarial, antiplatelet, antituberculosis, or antiviral activity was reported for 35 marine chemicals. An additional 20 marine compounds were shown to have significant effects on the cardiovascular and nervous system, and to possess anti-inflammatory or immunosuppressant properties. Finally, 23 marine compounds were reported to act on a variety of molecular targets and thus could potentially contribute to several pharmacologic classes. Thus, as in 1998 and 1999, during 2000 pharmacologic research with marine chemicals continued to contribute potentially novel chemical leads to the ongoing global search for therapeutic agents in the treatment of multiple disease categories.
189 citations
TL;DR: The cloning and functional characterization of 2 genes from salmon involved in the biosynthesis of HUFA are described, with the overall aim being to determine mechanisms for optimizing the use of vegetable oils in Atlantic salmon culture.
Abstract: Fish are the only major dietary source for humans of ω-3 highly unsaturated fatty acids (HUFAs) and with declining fisheries farmed fish such as Atlantic salmon (Salmo salar) constitute an increasing proportion of the fish in the human diet. However, the current high use of fish oils, derived from wild capture marine fisheries, in aquaculture feeds is not sustainable in the longer term and will constrain continuing growth of aquaculture activities. Greater understanding of how fish metabolize and biosynthesize HUFA may lead to more sustainable aquaculture diets. The study described here contributes to an effort to determine the molecular genetics of the HUFA biosynthetic pathway in salmon, with the overall aim being to determine mechanisms for optimizing the use of vegetable oils in Atlantic salmon culture. In this paper we describe the cloning and functional characterization of 2 genes from salmon involved in the biosynthesis of HUFA. A salmon desaturase complementary DNA, SalDes, was isolated that include an open reading frame of 1362 bp specifying a protein of 454 amino acids. The protein sequence includes all the characteristics of microsomal fatty acid desaturases, including 3 histidine boxes, 2 transmembrane regions, and an N-terminal cytochrome b5 domain containing a heme-binding motif similar to that of other fatty acid desaturases. Functional expression in the yeast Saccharomyces cerevisiae showed SalDes is predominantly an ω-3 δ5 desaturase, a key enzyme in the synthesis of eicosapentaenoic acid (20:5n-3) from α-linolenic acid (18:3n-3). The desaturase showed only low levels of δ6 activity toward C18 polyunsaturated fatty acids. In addition, a fatty acid elongase cDNA, SalElo, was isolated that included an open reading frame of 888 bp, specifying a protein of 295 amino acids. The protein sequence of SalElo included characteristics of microsomal fatty acid elongases, including a histidine box and a transmembrane region. Upon expression in yeast SalElo showed broad substrate specificity for polyunsaturated fatty acids with a range of chain lengths, with the rank order being C18 > C20 > C22. Thus this one polypeptide product displays all fatty acid elongase activities required for the biosynthesis of docosahexaenoic acid (22:6n-3) from 18:3n-3.
184 citations
TL;DR: Most distorted markers were deficient for homozygotes and closely linked to each other on the genetic map, suggesting the presence of major recessive deleterious genes in the Pacific oyster.
Abstract: Amplified fragment length polymorphisms (AFLPs) were used for genome mapping in the Pacific oyster Crassostrea gigas Thunberg. Seventeen selected primer combinations produced 1106 peaks, of which 384 (34.7%) were polymorphic in a backcross family. Among the polymorphic markers, 349 were segregating through either the female or the male parent. Chi-square analysis indicated that 255 (73.1%) of the markers segregated in a Mendelian ratio, and 94 (26.9%) showed significant (P < 0.05) segregation distortion. Separate genetic linkage maps were constructed for the female and male parents. The female framework map consisted of 119 markers in 11 linkage groups, spanning 1030.7 cM, with an average interval of 9.5 cM per marker. The male map contained 96 markers in 10 linkage groups, covering 758.4 cM, with 8.8 cM per marker. The estimated genome length of the Pacific oyster was 1258 cM for the female and 933 cM for the male, and the observed coverage was 82.0% for the female map and 81.3% for the male map. Most distorted markers were deficient for homozygotes and closely linked to each other on the genetic map, suggesting the presence of major recessive deleterious genes in the Pacific oyster.
162 citations
TL;DR: The preliminary results indicated that the majority of the identified microsatellites were polymorphic and, therefore, useful for genetic linkage mapping of catfish, and will set the foundation for comparative genome analysis in catfish.
Abstract: Gene-derived markers are pivotal to the analysis of genome structure, organization, and evolution and necessary for comparative genomics. However, gene-derived markers are relatively difficult to develop. This project utilized the genomic resources of channel catfish expressed sequence tags (ESTs) to identify simple sequence repeats (SSRs), or microsatellites. It took the advantage of ESTs for the establishment of gene identities, and of microsatellites for the acquisition of high polymorphism. When microsatellites are tagged to genes, the microsatellites can then be used as gene markers. A bioinformatic analysis of 43,033 ESTs identified 4855 ESTs containing microsatellites. Cluster analysis indicated that 1312 of these ESTs fell into 569 contigs, and the remaining 3534 ESTs were singletons. A total of 4103 unique microsatellite-containing genes were identified. The dinucleotide CA/TG and GA/TC pairs were the most abundant microsatellites. AT-rich microsatellite types were predominant among trinucleotide and tetranucleotide microsatellites, consistent with our earlier estimation that the catfish genome is highly AT-rich. Our preliminary results indicated that the majority of the identified microsatellites were polymorphic and, therefore, useful for genetic linkage mapping of catfish. Mapping of these gene-derived markers is under way, which will set the foundation for comparative genome analysis in catfish.
161 citations
TL;DR: Seasonally active antifouling activity of extracts of marine macroalgae against bacteria, fungi, diatoms, macroalgal spores, mussel phenoloxidase activity, and barnacle cypris larvae in bimonthly samples from the Bay of Concarneau is investigated.
Abstract: The antifouling activity of extracts (aqueous, ethanol, and dichloromethane) of 9 marine macroalgae against bacteria, fungi, diatoms, macroalgal spores, mussel phenoloxidase activity, and barnacle cypris larvae has been investigated in relation to season in bimonthly samples from the Bay of Concarneau (France). Of the extracts tested, 48.2% were active against at least one of the fouling organisms, and of these extracts, 31.2% were seasonally active with a peak of activity in summer corresponding to maximal values for water temperature, light intensity, and fouling pressure, and 17% were active throughout the year. This seasonal activity may be adaptive as it coincides with maximal fouling pressure in the Bay of Concarneau. Dichloromethane extracts of Rhodophyceae were the most active in the antifouling assays.
151 citations
TL;DR: The zebrafish enzyme was also able to elongate monounsaturated and saturated fatty acids, and thus demonstrates a greater level of promiscuity in terms of substrate use than any elongase enzyme described previously.
Abstract: Enzymes that increase the chain length of fatty acids are essential for biosynthesis of highly unsaturated fatty acids The gLELO gene encodes a protein involved in the elongation of polyunsaturated fatty acids in the fungus Mortierella alpina A search of the GenBank database identified several expressed sequence tag sequences, including one obtained from zebrafish (Danio rerio), with high similarity to gLELO The full-length transcript ZfELO, encoding a polypeptide of 291 amino acid residues, was isolated from zebrafish liver cDNA The predicted amino acid sequence of the open reading frame shared high similarity with the elongases of Caenorhabditis elegans and human When expressed in Saccharomyces cerevisiae, the zebrafish open reading frame conferred the ability to lengthen the chain of a range of C18, C20, and C22 polyunsaturated fatty acids, indicating not only that biosynthesis of 22:6n-3 from 18:3n-3 via a 24-carbon intermediate is feasible, but also that one elongase enzyme can perform all three elongation steps required The zebrafish enzyme was also able to elongate monounsaturated and saturated fatty acids, and thus demonstrates a greater level of promiscuity in terms of substrate use than any elongase enzyme described previously
147 citations
TL;DR: The sulfated exopolysaccharide p-KG03 exhibited impressive antiviral activity in vitro (EC50 = 26.9 µg/ml) against the encephalomyocarditis virus (EMCV) and is the first reported marine source of antiviral sulfated polysaccharides against EMCV.
Abstract: The sulfated exopolysaccharide p-KG03, which is produced by the marine microalga Gyrodinium impudicum strain KG03, exhibited impressive antiviral activity in vitro (EC50 = 26.9 µg/ml) against the encephalomyocarditis virus (EMCV). Depending on the p-KG03 concentration, the development of cytopathic effects in EMCV-infected HeLa cells was either inhibited completely or slowed. Moreover, p-KG03 did not show any cytotoxic effects on HeLa cells, even at concentrations up to 1000 µg/ml. The polysaccharide was purified by repeated precipitation in ethanol, followed by gel filtration. The p-KG03 polysaccharide had a molecular weight of 1.87 × 107, and was characterized as a homopolysaccharide of galactose with uronic acid (2.96% wt/wt) and sulfate groups (10.32% wt/wt). The biological activities of p-KG03 suggest that sulfated metabolites from marine organisms are a rich source of antiviral agents. This is the first reported marine source of antiviral sulfated polysaccharides against EMCV. The p-KG03 polysaccharide may be useful in the development of marine bioactive exopolysaccharide for biotechnological and pharmaceutical products.
118 citations
TL;DR: Various genes involved in signal transduction, innate immunity, and other processes have been uncovered in the subtractive libraries of Atlantic salmon experimentally infected with the pathogen Aeromonas salmonicida, the causative agent of furunculosis.
Abstract: In order to probe the interaction between an invading microorganism and its host, we have investigated differential gene expression in Atlantic salmon (Salmo salar) experimentally infected with the pathogen Aeromonas salmonicida, the causative agent of furunculosis. Subtractive cDNA libraries were constructed by suppression subtractive hybridization (SSH) from 3 immune-relevant tissues at 2 time points during the infection process. Both forward- and reverse-subtracted libraries were generated, and approximately 200 clones were sequenced from each library, giving a total of 1778 expressed sequence tags (ESTs), which were annotated according to functional categories and deposited in GenBank (BQ035314-BQ037059). Numerous genes involved in signal transduction, innate immunity, and other processes have been uncovered in the subtractive libraries. These include known acute-phase reactants, along with more novel genes encoding proteins such as tachylectin, hepcidin, precerebellin-like protein, O-methyltransferase, a putative saxitoxin-binding protein, and others. A subset of genes that were represented in the subtracted libraries was further analyzed by virtual Northern, or reverse transcription-polymerase chain reaction (RT-PCR) assays to verify their differential expression as a result of infection.
105 citations
TL;DR: The feasibility of single and multiple gene engineering of microorganisms to redirect their metabolism for improving H2 photoproduction using actual waste-derived substrates is demonstrated.
Abstract: Three differently metabolically engineered strains, 2 single PHA- and Hup- mutants and one double PHA-/Hup- mutant, of the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides RV, were constructed to improve a light-driven biohydrogen production process combined with the disposal of solid food wastes. These phenotypes were designed to abolish, singly or in combination, the competition of H2 photoproduction with polyhydroxyalkanoate (PHA) accumulation by inactivating PHA synthase activity, and with H2 recycling by abolishing the uptake hydrogenase enzyme. The performance of these mutants was compared with that of the wild-type strain in laboratory tests carried out in continuously fed photobioreactors using as substrates both synthetic media containing lactic acid and media from the acidogenic fermentation of actual fruit and vegetable wastes, containing mainly lactic acid, smaller amounts of acetic acia, and traces of higher volatile acids. With the lactic acid-based synthetic medium, the single Hup- and the double PHA-/Hup- mutants, but not the single PHA- mutant, exhibited increased rates of H2 photoproduction, about one third higher than that of the wild-type strain. With the food-waste-derived growth medium, only the single Hup- mutant showed higher rates of H2 production, but all 3 mutants sustained a longer-term H2 photoproduction phase than the wild-type strain, with the double mutant exhibiting overall the largest amount of H2 evolved. This work demonstrates the feasibility of single and multiple gene engineering of microorganisms to redirect their metabolism for improving H2 photoproduction using actual waste-derived substrates.
102 citations
TL;DR: Previous evidence, based on meiotic chromosome pairing and fluorescence in situ hybridization probes obtained through chromosome microdissection, that chromosome pair 1 is the sex chromosomes is confirmed.
Abstract: Gynogenetically produced XX and YY Nile tilapia (Oreochromis niloticus) and diploid control groups were screened for amplified fragment length polymorphisms (AFLPs) to search for sex-linked or sex-specific markers. Family-level bulked segregant analysis (XX and YY gynogenetic family pools) and individual screening (XX and YY gynogenetics and XX and XY control individuals) identified 3 Y-linked (OniY425, OniY382, OniY227) and one X-linked (OniX420) AFLP markers. OniX420 and OniY425 were shown to be allelic. Single locus polymerase chain reaction assays were developed for these markers. Tight linkage was demonstrated between the AFLP markers and the sex locus within the source families. However, these markers failed to consistently identify sex in unrelated individuals, indicating recombination between the markers and the sex-determining loci. O. niloticus bacterial artificial chromosome clones, containing the AFLP markers, hybridized to the long arm of chromosome 1. This confirmed previous evidence, based on meiotic chromosome pairing and fluorescence in situ hybridization probes obtained through chromosome microdissection, that chromosome pair 1 is the sex chromosomes.
97 citations
TL;DR: The X153 bacterium protected bivalve larvae against mortality, following experimental challenges with ichthyopathogenic Vibrio, and may be useful in aquaculture as a probiotic bacterium.
Abstract: A marine bacterium, X153, was isolated from a pebble collected at St. Anne du Portzic (France). By 16S ribosomal DNA gene sequence analysis, X153 strain was identified as a Pseudoalteromonas sp. close to P. piscicida. The crude culture of X153 was highly active against human pathogenic strains involved in dermatologic diseases, and marine bacteria including various ichthyopathogenic Vibrio strains. The active substance occurred both in bacterial cells and in culture supernatant. An antimicrobial protein was purified to homogeneity by a 4-step procedure using size-exclusion and ion-exchange chromatography. The highly purified P-153 protein is anionic, and sodium dodecylsulfate polyacrylamide gel electrophoresis gives an apparent molecular mass of 87 kDa. The X153 bacterium protected bivalve larvae against mortality, following experimental challenges with ichthyopathogenic Vibrio. Pseudoalteromonas sp. X153 may be useful in aquaculture as a probiotic bacterium.
TL;DR: Analysis of genetic variation and population structure of wild white shrimp from 4 geographic locations from Mexico to Panama showed that the white shrimp population is structured into subpopulations, however, population differentiation does not follow an isolation-by-distance model.
Abstract: Genetic variation and population structure of wild white shrimp (Litopenaeus vannamei) from 4 geographic locations from Mexico to Panama were investigated using 5 microsatellite DNA loci. The genetic diversity between populations was indicated by the mean number of alleles per locus and mean observed heterozygosity, which ranged from 7.4 to 8.6 and from 0.241 to 0.388, respectively. Significant departures from Hardy-Weinberg equilibrium were found at most locations at each locus, with the exception Guatemala at Pvan0013, and were caused by high heterozygote deficiencies. Genetic differences between localities were detected by pairwise comparison based on allelic and genotypic frequencies, with the exception of locus Pvan1003. Significant pairwise F
ST values between locations and total F
ST showed that the white shrimp population is structured into subpopulations. However, population differentiation does not follow an isolation-by-distance model. Knowledge of the genetic diversity and structure of L.vannamei populations will be of interest for aquaculture and fisheries management to utilize and preserve aquatic biodiversity.
TL;DR: The results presented here indicate that a photoprotective function of GFP-like proteins is unlikely in the examined anthozoa species.
Abstract: We screened nonbioluminescent, azooxanthellate cnidaria as potential sources for advanced marker proteins and succeeded in cloning a tetrameric green fluorescent protein (GFP) from the tentacles of Cerianthus membranaceus. The fluorescence of this protein (cmFP512) is characterized by excitation maximum at 503 nm, emission maximum at 512 nm, extinction coefficient of 58,800 M−1 cm−1, quantum yield of 0.66, and fluorescence lifetime of 2.4 ns. The chromophore is formed from the tripeptide Gln-Tyr-Gly. The amino acid sequence of this protein shares 17.8% identical residues with GFP from Aequorea victoria. Weak interactions between the subunits of the tetramer make cmFP512 a promising lead structure for the generation of monomeric variants of fluorescent proteins. Both red fluorescent proteins and nonfluorescent proteins of the GFP family were also purified from tissue homogenates of Adamsia palliata and Calliactis parasitica. The results presented here indicate that a photoprotective function of GFP-like proteins is unlikely in the examined anthozoa species.
TL;DR: A 3D-aggregate primmorph culture system was established and it was proved that one bioactive compound, avarol/avarone, is produced by the sponge Dysidea avara, and another promising way to utilize the bioactive potential of the microorganisms is the cloning and heterologous expression of enzymes involved in secondary metabolism, such as the polyketide synthases.
Abstract: Sponges (phylum Porifera) are sessile marine filter feeders that have developed efficient defense mechanisms against foreign attackers such as viruses, bacteria, or eukaryotic organisms. Protected by a highly complex immune system, as well as by the capacity to produce efficient antiviral compounds (e.g., nucleoside analogues), antimicrobial compounds (e.g., polyketides), and cytostatic compounds (e.g., avarol), they have not become extinct during the last 600 million years. It can be assumed that during this long period of time, bacteria and microorganisms coevolved with sponges, and thus acquired a complex common metabolism. It is suggested that (at least) some of the bioactive secondary metabolites isolated from sponges are produced by functional enzyme clusters, which originated from the sponges and their associated microorganisms. As a consequence, both the host cells and the microorganisms lost the ability to grow independently from each other. Therefore, it was—until recently—impossible to culture sponge cells in vitro. Also the predominant number of “symbiotic bacteria” proved to be nonculturable. In order to exploit the bioactive potential of both the sponge and the “symbionts,” a 3D-aggregate primmorph culture system was established; also it was proved that one bioactive compound, avarol/avarone, is produced by the sponge Dysidea avara. Another promising way to utilize the bioactive potential of the microorganisms is the cloning and heterologous expression of enzymes involved in secondary metabolism, such as the polyketide synthases.
TL;DR: This protocol clearly resolves the taxonomic ambiguities of closely related algal species (such as Alexandrium and Cochlodinium), and it constitutes a significant breakthrough for the molecular analysis of nonculturable dinoflagellate species.
Abstract: A simplified technique was developed for DNA sequence-based diagnosis of harmful dinoflagellate species. This protocol integrates procedures for DNA extraction and polymerase chain reaction (PCR) amplification into a single tube. DNA sequencing reactions were performed directly, using unpurified PCR products as the DNA template for subsequent sequencing reactions. PCR reactions using DNA extracted from single cells of Cocodinium polykrikoides and Alexandrium catenella successfully amplified the target ribosomal DNA regions. DNA sequencing of the unpurified PCR products showed that DNA sequences corresponded to the expected locus of ribosomal DNA regions of both A. catenella and C. polykrikoides (each zero genetic distance and 100% sequence similarity). Using the protocol described in this article, there was little DNA loss during the purification step, and the technique was found to be rapid and inexpensive. This protocol clearly resolves the taxonomic ambiguities of closely related algal species (such as Alexandrium and Cochlodinium), and it constitutes a significant breakthrough for the molecular analysis of nonculturable dinoflagellate species.
TL;DR: Variation in the NADH subunit coding regions is adequate to resolve phylogenetic relationships among families and some genera of Octocorallia, but insufficient for most species - or population-level studies.
Abstract: Low rates of evolution in cnidarian mitochondrial genes such as COI and 16S rDNA have hindered molecular systematic studies in this important invertebrate group. We sequenced fragments of 3 mitochondrial protein-coding genes (NADH dehydrogenase subunits ND2, ND3 and ND6) as well as the COI-COII intergenic spacer, the longest noncoding region found in the octocoral mitochondrial genome, to determine if any of these regions contain levels of variation sufficient for reconstruction of phylogenetic relationships among genera of the anthozoan subclass Octocorallia. Within and between the soft coral families Alcyoniidae and Xeniidae, sequence divergence in the genes ND2 (539 bp), ND3 (102 bp), and ND6 (444 bp) ranged from 0.5% to 12%, with the greatest pairwise distances between the 2 families. The COI-COII intergenic spacer varied in length from 106 to 122 bp, and pairwise sequence divergence values ranged from 0% to 20.4%. Phylogenetic trees constructed using each region separately were poorly resolved. Better phylogenetic resolution was obtained in a combined analysis using all 3 protein-coding regions (1085 bp total). Although relationships among some pairs of species and genera were well supported in the combined analysis, the base of the alcyoniid family tree remained an unresolved polytomy. We conclude that variation in the NADH subunit coding regions is adequate to resolve phylogenetic relationships among families and some genera of Octocorallia, but insufficient for most species - or population-level studies. Although the COI-COII intergenic spacer exhibits greater variability than the protein-coding regions and may contain useful species-specific markers, its short length limits its phylogenetic utility.
TL;DR: This work characterized a topographic and time-dependent zonation of matrix proteins involved in shell biomineralization in the mantle of Haliotis, and provided information about the source of shell matrix macromolecule production, within the external epithelium of the mantle.
Abstract: Organic matrix from molluscan shells has the potential to regulate calcium carbonate deposition and crystallization. Control of crystal growth thus seems to depend on control of matrix protein secretion or activation processes in the mantle cells, about which little is known. Biomineralization is a highly orchestrated biological process. The aim of this work was to provide information about the source of shell matrix macromolecule production, within the external epithelium of the mantle. An in vivo approach was chosen to describe the histologic changes in the outer epithelium and in blood sinus distribution, associated with mantle cells implicated in shell matrix production. Our results characterized a topographic and time-dependent zonation of matrix proteins involved in shell biomineralization in the mantle of Haliotis.
TL;DR: Direct sequence evidence suggests that these two isoforms come from a Chh gene transcribed in an alternative splicing manner, and the common organization of Chh genes between palaemonid, brachyuran, and astacus crustaceans suggests that the ChH gene has a 4-exon structure in these species.
Abstract: A full-length chh cDNA was cloned from the eyestalk of Macrobrachium rosenbergii. The 991-bp cDNA contains an open reading frame of 408 bp that encodes the prepro-CHH. The tissue-specific expression pattern was examined by reverse transcriptase-polymerase chain reaction. Positive signals were detected in the eyestalk, heart, gills, antennal glands, and thoracic ganglion but not in muscle and hepatopancreas. However, two types of products were observed. The nucleotide sequences revealed the existence of 2 chh transcripts, named chh and chh-l, respectively. Direct sequence evidence suggests that these two isoforms come from a Chh gene transcribed in an alternative splicing manner. The Mar-Chh gene consists of 4 exons. The eyestalk transcript (chh) contains exons I, II, and IV, whereas the chh-l transcript in heart, gills, antennal glands, and thoracic ganglion contains all 4 exons. The appearance of exon III in chh-l cDNA changes the sequence content in the latter half of the mature peptide, starting within the codon of the 40th residue, arginine. The amino acid sequence deduced from exon III matched no homologue in public protein databases, while the 2 cysteine residues in this segment preserved the positional conservation characters of CHH neuropeptide family members. The common organization of Chh genes between palaemonid, brachyuran, and astacus crustaceans suggests that the Chh gene has a 4-exon structure in these species.
TL;DR: The catalog of ESTs reported herein represents a significant increase in the limited sequence information currently available for E. huxleyi and should make the coccolithophorid more accessible to powerful genomics and postgenomics technologies.
Abstract: An expressed sequence tag (EST) approach was used to investigate gene expression in the unicelluar marine alga Emiliania huxleyi. We randomly selected 3000 EST sequences from a cDNA library of transcripts expressed under conditions promoting coccolithogenesis. Cluster analysis and contig assembly resulted in a unigene set of approximately 1523 ESTs. Only 36% of the unique sequences exhibited significant homology to sequences in GenBank. Of particular interest were the numerous transcripts with homology to sequences associated with sexual reproduction and calcium homeostasis in other unicellular and multicellular organisms. The majority of ESTs (64%) had little or no significant sequence homology to entries in GenBank, suggesting a potential for further novel gene discovery. The catalog of ESTs reported herein represents a significant increase in the limited sequence information currently available for E. huxleyi and should make the coccolithophorid more accessible to powerful genomics and postgenomics technologies.
TL;DR: Type and activities of lobster digestive enzymes constitute background information to study the digestive abilities of the organism further, and will lead to understanding nutritional needs and feeding ecology, mainly because decapods display unique morphologic, metabolic, and behavioral changes during their life cycle.
Abstract: Enzymes responsible for digestion of food protein were evaluated and characterized in red lobster (Panulirus interruptus) Several tissues, organs, and body fluids were analyzed The same composition of proteases was found in gastric juice, midgut gland, and intestinal contents Using specific substrates and inhibitors, we indentified several isotrypsins and isochymotrypsins by gel electrophoresis Protease activity was found at pH 3 and reduced by using pepstatin A Operational variables of enzymes were characterized for management of future studies and potential biotechnologies Types and activities of lobster digestive enzymes constitute background information to study the digestive abilities of the organism further, and will lead to understanding nutritional needs and feeding ecology, mainly because decapods display unique morphologic, metabolic, and behavioral changes during their life cycle Also, such enzymes become alternative tools for use in biotechnologies
TL;DR: It is suggested that real-time PCR can be a powerful tool for detecting Japanese eel eggs and newly hatched larvae immediately after onboard sampling during research cruises and will allow targeted sampling efforts to occur rapidly in response to any positive onboard identification of the eggs and larvae of this species.
Abstract: To compensate for the limited number of morphological characteristics of fish eggs and larvae, we established a convenient and robust method of species identification for eggs of the Japanese eel (Anguilla japonica) using a real-time polymerase chain reaction (PCR) that can be performed onboard research ships at sea. A total of about 1.2 kbp of the mitochondrial 16S ribosomal RNA gene sequences from all species of Anguilla and 3 other anguilliform species were compared to design specific primer pairs and a probe for A. japonica. This real-time PCR amplification was conducted for a total of 44 specimens including A. japonica, A. marmorata, A. bicolor pacifica, and 6 other anguilliform species. Immediate PCR amplification was only observed in A. japonica. We then tested this method under onboard conditions and obtained the same result as had been produced in the laboratory. These results suggest that real-time PCR can be a powerful tool for detecting Japanese eel eggs and newly hatched larvae immediately after onboard sampling during research cruises and will allow targeted sampling efforts to occur rapidly in response to any positive onboard identification of the eggs and larvae of this species.
TL;DR: Two strains subjected to long-term selection for disease resistance shared frequency shifts at a few loci, which deserve further analysis to determine if they are linked to disease-resistance genes.
Abstract: Amplified fragment length polymorphisms (AFLPs) and microsatellite markers were used to examine genetic variation and divergence in 4 selected strains (DBH, NEH, FMF, and CTS) and 1 wild population (DBW) of the eastern oyster Crassostrea virginica Gmelin. Eighty-six AFLP markers (from 3 primer pairs) and 5 microsatellite loci were used for the analysis of 30 oysters from each of the 5 populations. Microsatellite loci were considerably more variable than AFLPs. The observed heterozygosity ranged from 0.560 to 0.640 across populations for microsatellites, and from 0.186 to 0.207 for AFLPs. Both Fst and phiPT of microsatellite data and phiPT statistics of AFLP data revealed significant divergence between all pairs of populations. There was no significant reduction in heterozygosity in all 4 selected strains; however, the number of alleles per locus was considerably lower in the selected strains than in the wild population. Two strains subjected to long-term selection for disease resistance shared frequency shifts at a few loci, which deserve further analysis to determine if they are linked to disease-resistance genes.
TL;DR: An approach for sequencing the entire mitochondrial genomes (mitogenomes) of decapod crustaceans using 79 newly designed and 7 published polymerase chain reaction (PCR) primers is described, which allows a more rapid determination of decapd mitogenomic sequences.
Abstract: An approach for sequencing the entire mitochondrial genomes (mitogenomes) of decapod crustaceans using 79 newly designed and 7 published polymerase chain reaction (PCR) primers is described. The approach comprises the following steps: (1) the entire mitogenome is amplified in 2 or 3 long PCRs; (2) the 86 primers are used in different combinations to amplify contiguous, overlapping short segments of the entire mitogenome with the diluted long PCR products as templates; (3) direct cycle sequencing is conducted using the short PCR products. This strategy allows a more rapid determination of decapod mitogenomic sequences than a traditional method using cloned mitochondrial DNA and primer walking strategy. As a practical example, the mitogenomic sequence for a kuruma prawn Marsupenaeus japonicus (Crustacea: Decapoda), was determined using the PCR-based approach.
TL;DR: Two new and 8 known cyclic dipeptides were isolated and characterized from the fermentation broth of SDC-1, which was identified by 16S ribosomal RNA sequence analysis as an α-Proteobacterium of the genus Ruegeria.
Abstract: From cell cultures of Suberites domuncula was isolated a bacterial strain, SDC-1, which was identified by 16S ribosomal RNA sequence analysis as an α-Proteobacterium of the genus Ruegeria. The occurrence of the strain in sponge cell culture could be explained by its resistance to the antibiotics used in the isolation of sponge cell cultures or by the preservation of SDC-1 by host sponge cells. The fatty acid composition of SDC-1 is characterized by branched C-12 methyl fatty acids. Two new and 8 known cyclic dipeptides were isolated and characterized from the fermentation broth of SDC-1. Cyclodipeptides are one of the families of cell-cell signaling compounds and may have some role to play in sponge-bacteria interactions.
TL;DR: An agar culture medium plate containing antibiotics and a giant clam tissue homogenate is developed and used to isolate two new Symbiodinium strains from two molluscan hosts, each of which hosted two different Symbiod inium strains belonging to SymbiodInium C and D, respectively.
Abstract: Recent molecular biological studies have revealed that some photosymbiotic invertebrates dwelling in coral reefs host several genetically different dinoflagellates, Symbiodinium species, as symbionts. However, little is known about the difference in physiologic characteristics among these symbionts living in a single host, because some Symbiodinium strains are difficult to culture in vitro. To isolate some of these Symbiodinium strains, we have developed an agar culture medium plate containing antibiotics and a giant clam tissue homogenate. Using-this medium we isolated two new Symbiodinium strains from two molluscan hosts, Tridacna crocea and Pteraeolidia ianthina, each of which hosted two different Symbiodinium strains belonging to Symbiodinium C and D, respectively. The tissue homogenate was essential for the growth of Symbiodinium D. Although it was not essential for the growth of Symbiodinium C, it did stimulate the initial growth. For the isolation of some Symbiodinium strains, isolation medium containing host homogenate is effective.
TL;DR: The isolation and the molecular characterization of silicatein from spicules of Petrosia ficiformis, and the development of a cellular model (primmorphs) suitable for in vitro studies of silicatesin gene regulation are described.
Abstract: In some sponges peculiar proteins called silicateins catalyze silica polymerization in ordered structures, and their study is of high interest for possible biotechnological applications in the nanostructure industry. In this work we describe the isolation and the molecular characterization of silicatein from spicules of Petrosia ficiformis, a common Mediterranean sponge, and the development of a cellular model (primmorphs) suitable for in vitro studies of silicatein gene regulation. The spicule of P. ficiformis contains an axial filament composed of 2 insoluble proteins, of 30 and 23 kDa. The 23-kDa protein was characterized, and the full-length cDNA was cloned. The putative amino acid sequence has high homology with previously described silicateins from other sponge species and also is very similar to cathepsins, a cystein protease family. Finally, P. ficiformis primmorphs express the silicatein gene, suggesting that they should be a good model for biosilicification studies.
TL;DR: All population analyses were consistent with the hypothesis that red snapper constitute a single, panmictic population over the sampled range, and a ubiquitous, predominant haplotype appeared to be evolutionarily recent, in contrast to previous findings based on restriction fragment length polymorphism data.
Abstract: The mitochondrial DNA control regions of red snapper (Lutjanus campechanus) from the Gulf of Mexico (n = 140) and Atlantic coast of Florida (n = 35) were sequenced to generate a prestocking genetic baseline for planned stock enhancement. Intrasample haplotype and nucleotide diversities ranged from 0.94 to 1.00 and 1.8% to 2.5%, respectively. All population analyses were consistent with the hypothesis that red snapper constitute a single, panmictic population over the sampled range. A ubiquitous, predominant haplotype, shared by 23% of the specimens, appeared to be evolutionarily recent, in contrast to previous findings based on restriction fragment length polymorphism data. Tajima’s D values were suggestive of a recent bottleneck. Mismatch distributions from Gulf samples were smooth and unimodal, characteristic of recent population expansion. However, the Atlantic sample exhibited a comparatively broader, possibly multimodal distribution, suggestive of a more stable population history. Additional control-region data may clarify potentially disparate demographic histories of Gulf and Atlantic snapper.
TL;DR: A marine bacterial strain that degraded fucoidan from Kjellmaniella crassifolia (class Phaeophyceae, order Laminariales, family Laminariaceae) was isolated in the laboratory and produced a type of extracellularfucoidanase, an endosulfated fucan-digesting enzyme.
Abstract: A marine bacterial strain that degraded fucoidan from Kjellmaniella crassifolia (class Phaeophyceae, order Laminariales, family Laminariaceae) was isolated in our laboratory. The strain was gram-negative, ubiquinone 8 was the predominant respiratory quinone, and the GC-content of its genomic DNA was 36%. The cells of the strain were rod-shaped (2.0 microm long x 1.0 microm wide), and each cell was motile by means of one polar flagellum. Phylogenetic analysis of its 16S ribosomal DNA sequence indicated that it was a member of the family Alteromonadaceae. It produced a type of extracellular fucoidanase, an endosulfated fucan-digesting enzyme. The enzyme was purified with 3500-fold purity at 12.0% yield. Optimum conditions for the enzyme reaction were approximately pH 6.5 to 8.0 and temperature 30 degrees to 35 degrees C. The enzyme was activated by calcium ions, and maximum activity was observed in the presence of greater than 30 mM calcium ion.
TL;DR: Genetic diversity in a shrimp-breeding program was monitored for 2 generations by microsatellite DNA markers to establish levels of variation and proceed with a selection program.
Abstract: Genetic diversity in a shrimp-breeding program was monitored for 2 generations by microsatellite DNA markers (Pvan1578 and Pvan1815) to establish levels of variation and proceed with a selection program. An increase in the number and frequencies of some alleles in both microsatellite loci from G0 to G2 was induced by foreign sire contributions. Most common alleles and high heterozygosities (around 70% in both loci) were maintained through the generations, indicating that there had not been a significant loss of genetic variability in the breeding program. However, when compared with variability in other wild and cultured stocks, the presence of 4 main alleles at both loci may be an indication that a certain reduction in variability already was present in the line used as founder stock (G0). Therefore, it is recommended that additional genetic variability be introduced to the breeding stock by crossing it with a different line.
TL;DR: A modified transformation strategy using a natural Tn5 transposon, transposase, and cation liposome complex by electroporation is applied to improve the transformation efficiency for Spirulina platensis strain C1 (Arthrospira sp. PCC9438).
Abstract: Spirulina platensis is one of the most commercially important species of microalgae. Thus, it is an attractive candidate for genetic manipulation and the development of novel practical applications. However, this process is hampered by the absence of a stable gene transfer system, specifically the limited number of suitable vectors and transformation methods available for this organism. Artificial transposon systems developed by extracting the essential elements from natural transposons have been extensively studied, and recently a mutated transposase and transposon system was reported to improve transformation efficiency by electroporation. We applied a modified transformation strategy using a natural Tn5 transposon, transposase, and cation liposome complex by electroporation to improve the transformation efficiency for Spirulina platensis strain C1 (Arthrospira sp. PCC9438). Aggregation of cells became visible after 3 weeks during 2.0 microg/ml chloramphenicol selection, and growth continued for more than 12 months. Transfected chloramphenicol acetyltransferase (CAT) genes were detected in the genomic DNA by Southern hybridization. Transformed cells demonstrated CAT activity, but non-transformed cells did not.