Jadomycins derived from the assimilation and incorporation of norvaline and norleucine.

Streptomyces venezuelae ISP5230 is recognized for the production of chloramphenicol and the jadomycin family of natural products. The jadomycins are angucycline natural products containing a unique oxazolone ring incorporating an amino acid present in the minimal culture media. Substitution of different amino acids results in products of varying biological activity. Analysis of cultures of S. venezuelae ISP5230 incubated with l- and d-norvaline and l- and d-norleucine indicated that only the d-configured amino acids were incorporated into the natural products. Subsequently, jadomycin DNV and jadomycin DNL were isolated and characterized (titers 4 and 9 mg L(-1), respectively). The compounds were evaluated in the National Cancer Institute cell line cancer growth inhibition and cytotoxicity screens, for antimicrobial activity against selected Gram-positive and Gram-negative bacteria, and as DNA-cleavage agents in vitro.

A novel target-specific, salt-resistant antimicrobial peptide against the cariogenic pathogen Streptococcus mutans.

In this study, we constructed and evaluated a target-specific, salt-resistant antimicrobial peptide (AMP) that selectively targeted Streptococcus mutans, a leading cariogenic pathogen. The rationale for creating such a peptide was based on the addition of a targeting domain of S. mutans ComC signaling peptide pheromone (CSP) to a killing domain consisting of a portion of the marine-derived, broad-spectrum AMP pleurocidin to generate a target-specific AMP. Here, we report the results of our assessment of such fusion peptides against S. mutans and two closely related species. The results showed that nearly 95% of S. mutans cells lost viability following exposure to fusion peptide IMB-2 (5.65 µM) for 15 min. In contrast, only 20% of S. sanguinis or S. gordonii cells were killed following the same exposure. Similar results were also observed in dual-species mixed cultures of S. mutans with S. sanguinis or S. gordonii. The peptide-guided killing was further confirmed in S. mutans biofilms and was shown to be dose dependent. An S. mutans mutant defective in the CSP receptor retained 60% survival following exposure to IMB-2, suggesting that the targeted peptide predominantly bound to the CSP receptor to mediate killing in the wild-type strain. Our work confirmed that IMB-2 retained its activity in the presence of physiological or higher salt concentrations. In particular, the fusion peptide showed a synergistic killing effect on S. mutans with a preventive dose of NaF. In addition, IMB-2 was relatively stable in the presence of saliva containing 1 mM EDTA and did not cause any hemolysis. We also found that replacement of serine-14 by histidine improved its activity at lower pH. Because of its effectiveness, salt resistance, and minimal toxicity to host cells, this novel target-specific peptide shows promise for future development as an anticaries agent.

The zebrafish embryo as a tool for screening and characterizing pleurocidin host-defense peptides as anti-cancer agents.

The emergence of multidrug-resistant cancers and the lack of targeted therapies for many cancers underscore an unmet need for new therapeutics with novel modes of action towards cancer cells. Host-defense peptides often exhibit selective cytotoxicity towards cancer cells and show potential as anti-cancer therapeutics. Here, we screen 26 naturally occurring variants of the peptide pleurocidin for cytotoxic and anti-cancer activities, and investigate the underlying mechanism of action. Cytotoxicities were assessed in vitro using cell-based assays and in vivo using zebrafish embryos. Morphological changes were assessed by both transmission and scanning electron microscopy, and functional assays were performed on zebrafish embryos to investigate the mechanism of cell death. A total of 14 peptides were virtually inactive against HL60 human leukemia cells, whereas 12 caused >50% death at ≤32 µg/ml. Morphological changes characteristic of oncosis were evident by electron microscopy after only 1 minute of treatment with 32 µg/ml of variant NRC-03. Only two peptides were hemolytic. Four peptides showed no toxicity towards zebrafish embryos at the highest concentration tested (25 µM; ∼64 µg/ml) and one peptide was highly toxic, killing 4-hour-post-fertilization (hpf) embryos immediately after exposure to 1 µM peptide. Four other peptides killed embryos after 24 hours of exposure at 1 µM. Most peptides caused mortality at one or more developmental stages only after continuous exposure (24 hours) with higher lethal doses (≥5 µM). Pleurocidin NRC-03 bound to embryos and induced the release of superoxide, caused an increase in the number of TUNEL-positive nuclei, and caused membrane damage and the loss of embryonic epithelial integrity, marked by the exclusion of cells from the outer epithelium and the appearance of F-actin within the circumferential cells of the repair site. Our results indicate that specific pleurocidin variants are attractive cancer-selective agents that selectively induce cell death in target cells but leave non-target cells such as erythrocytes and non-transformed cells unaffected.

Antimicrobial activities of jadomycin B and structurally related analogues.

Natural products are leads for new antibiotics as a result of their structural complexity and diversity. We have isolated a series of structurally related polyketide-derived natural products from Streptomyces venezuelae ISP5230. The most active of these jadomycin analogues showed good activity against a variety of staphylococci, including methicillin-resistant Staphylococcus aureus.

Translational machinery of senegalese sole (Solea senegalensis Kaup) and Atlantic halibut (Hippoglossus hippoglossus L.): comparative sequence analysis of the complete set of 60s ribosomal proteins and their expression.

Ribosomal proteins (RPs) comprise a large set of highly evolutionarily conserved proteins that are often over-represented in complementary DNA libraries. They have become very useful markers in comparative genomics, genome evolution, and phylogenetic studies across taxa. In this study, we report the sequences of the complete set of 60S RPs in Senegalese sole (Solea senegalensis) and Atlantic halibut (Hippoglossus hippoglossus), two commercially important flatfish species. Amino-acid sequence comparisons of the encoded proteins showed a high similarity both between these two flatfish species and with respect to other fish and human counterparts. Expressed sequence tag analysis revealed the existence of paralogous genes for RPL3, RPL7, RPL41, and RPLP2 in Atlantic halibut and RPL13a in Senegalese sole as well as RPL19 and RPL22 in both species. Phylogenetic analysis of paralogs revealed distinct evolutionary histories for each RP in agreement with three rounds of genome duplications and lineage-specific duplications during flatfish evolution. Steady-state transcript levels for RPL19 and RPL22 RPs were quantitated during larval development and in different tissues of sole and halibut using a real-time polymerase chain reaction approach. All paralogs were expressed ubiquitously although at different levels in different tissues. Most RP transcripts increased coordinately after larval first-feeding in both species but decreased progressively during the metamorphic process. In all cases, expression profiles and transcript levels of orthologous genes in Senegalese sole and Atlantic halibut were highly congruent. The genomic resources and knowledge developed in this survey will be useful for the study of Pleuronectiformes evolution.

Ontogenetic and tissue-specific expression of preproghrelin in the Atlantic halibut, Hippoglossus hippoglossus L.

Ghrelin is a conserved vertebrate hormone that affects both GH release and appetite. We have cloned and characterized Atlantic halibut preproghrelin cDNA and examined for the first time preproghrelin expression during fish larval development using quantitative real-time PCR. In addition, cellular sites of expression in larvae and tissue-specific expression in 3-year-old halibut were studied. A full-length cDNA for preproghrelin was isolated from halibut stomach tissue. The 899 bp cDNA encodes an open reading frame of 105 amino acids that is comprised of a signal peptide and two peptides with high similarity to ghrelin and obestatin. The deduced amino acid sequence of halibut ghrelin peptide (GSSFLSPSHKPPKGKPPRA) shows significant conservation relative to other teleostean sequences and is identical to human ghrelin for the first seven amino acids of the sequence. The putative obestatin peptide is well-conserved among fishes but shares limited similarity with its human counterpart. Expression of ghrelin was localized to two different cell types in the stomach of larval halibut by in situ hybridization. However, sensitive PCR assays on tissues collected from 3-year-old fish additionally identified ghrelin transcripts in pyloric caecae, intestine, and in immature ovary and testis. Ontogenetic studies detected ghrelin expression prior to exogenous feeding during larval development (hatching and mouth-opening stages) with increased expression occurring through metamorphosis. This increase was pronounced during climax metamorphosis and coincided with stomach differentiation. Patterns of preproghrelin expression suggest that ghrelin has important roles during and after larval development in halibut, and that ghrelin is associated with digestive and gonadal tissues in this teleost.

The early response of Atlantic salmon (Salmo salar) macrophages exposed in vitro to Aeromonas salmonicida cultured in broth and in fish.

Aeromonas salmonicida is a fish pathogen that causes furunculosis. Virulent strains of this bacterium are able to infect salmonid macrophages and survive within them, although mechanisms favouring intracellular survival are not completely understood. It is known that A. salmonicida cultured in vivo in the peritoneal cavity of the host undergoes changes in gene expression and surface architecture compared with cultures grown in vitro in broth. Therefore, in this study, the early macrophage responses to A. salmonicida grown in vivo and in vitro were compared. Macrophage-enriched cell preparations from head kidney of Atlantic salmon (Salmo salar) were infected in vitro in 96-well microtitre dishes and changes in gene expression during the infection process were monitored using a custom Atlantic salmon cDNA microarray. A. salmonicida cultures grown in tryptic soy broth and in peritoneal implants were used to infect the macrophages. The macrophages were harvested at 0.5, 1.0 and 2.0h after addition of the bacteria to the medium. Significant changes in gene expression were evident by microarray analysis at 2.0h post-infection in macrophages infected with broth-grown and implant-grown bacteria; however, qPCR analysis revealed earlier up-regulation of JunB and TNF-alpha in macrophages exposed to the implant-grown bacteria. Up-regulation of those genes and others is consistent with the effects of extracellular products of aeromonad bacteria on macrophages and also suggests initiation of the innate immune response.

Comparative sequence analysis of the complete set of 40S ribosomal proteins in the Senegalese sole (Solea senegalensis Kaup) and Atlantic halibut (Hippoglossus hippoglossus L.) (Teleostei: Pleuronectiformes): phylogeny and tissue- and development-specific expression.

BACKGROUND: Ribosomal proteins (RPs) are key components of ribosomes, the cellular organelle responsible for protein biosynthesis in cells. Their levels can vary as a function of organism growth and development; however, some RPs have been associated with other cellular processes or extraribosomal functions. Their high representation in cDNA libraries has resulted in the increase of RP sequences available from different organisms and their proposal as appropriate molecular markers for phylogenetic analysis. RESULTS: The development of large-scale genomics of Senegalese sole (Solea senegalensis) and Atlantic halibut (Hippoglossus hippoglossus), two commercially important flatfish species, has made possible the identification and systematic analysis of the complete set of RP sequences for the small (40S) ribosome subunit. Amino acid sequence comparisons showed a high similarity both between these two flatfish species and with respect to other fish and human. EST analysis revealed the existence of two and four RPS27 genes in Senegalese sole and Atlantic halibut, respectively. Phylogenetic analysis clustered RPS27 in two separate clades with their fish and mammalian counterparts. Steady-state transcript levels for eight RPs (RPS2, RPS3a, RPS15, RPS27-1, RPS27-2, RPS27a, RPS28, and RPS29) in sole were quantitated during larval development and in tissues, using a real-time PCR approach. All eight RPs exhibited different expression patterns in tissues with the lowest levels in brain. On the contrary, RP transcripts increased co-ordinately after first larval feeding reducing progressively during the metamorphic process. CONCLUSION: The genomic resources and knowledge developed in this survey will provide new insights into the evolution of Pleuronectiformes. Expression data will contribute to a better understanding of RP functions in fish, especially the mechanisms that govern growth and development in larvae, with implications in aquaculture.

Comprehensive EST analysis of Atlantic halibut (Hippoglossus hippoglossus), a commercially relevant aquaculture species.

BACKGROUND: An essential first step in the genomic characterisation of a new species, in this case Atlantic halibut (Hippoglossus hippoglossus), is the generation of EST information. This forms the basis for subsequent microarray design, SNP detection and the placement of novel markers on genetic linkage maps. RESULTS: Normalised directional cDNA libraries were constructed from five different larval stages (hatching, mouth-opening, midway to metamorphosis, premetamorphosis, and post-metamorphosis) and eight different adult tissues (testis, ovary, liver, head kidney, spleen, skin, gill, and intestine). Recombination efficiency of the libraries ranged from 91-98% and insert size averaged 1.4 kb. Approximately 1000 clones were sequenced from the 5'-end of each library and after trimming, 12675 good sequences were obtained. Redundancy within each library was very low and assembly of the entire EST collection into contigs resulted in 7738 unique sequences of which 6722 (87%) had matches in Genbank. Removal of ESTs and contigs that originated from bacteria or food organisms resulted in a total of 7710 unique halibut sequences. CONCLUSION: A Unigene collection of 7710 functionally annotated ESTs has been assembled from Atlantic halibut. These have been incorporated into a publicly available, searchable database and form the basis for an oligonucleotide microarray that can be used as a tool to study gene expression in this economically important aquacultured fish.

Exploring the diversity of marine-derived fungal polyketide synthases.

Using an approach based on polymerase chain reaction (PCR), we examined the diversity of polyketide synthase (PKS) genes present in 160 marine fungal isolates, representing 142 species. We obtained ketosynthase (KS) domain PCR products from 99 fungal isolates, representing Dothideomycetes, Sordariomycetes, Eurotiomycetes, and incertae sedis. Sequence similarity searches and phylogenetic analysis of 29 marine partial-KS-encoding sequences revealed domains predicted to encode reducing, nonreducing, and 6-methylsalicylic acid PKSs. Bioinformatic analysis of an alignment of the KS sequences from marine-derived fungi revealed no unique motifs in this region. However, several specificity-determining positions were apparent between fungal 6-methylsalicylic acid PKSs as compared with either reducing or nonreducing PKSs. Evaluation of these positions in the context of a modelled three-dimensional protein structure highlighted their potential use as PKS classification markers. Evaluating primer-binding sites was necessary to obtain KS domain fragments from putative PKSs while maintaining a level of sequence information adequate to properly classify and characterize them.

Isolation and characterization of cDNA sequences of L-gulono-gamma-lactone oxidase, a key enzyme for biosynthesis of ascorbic acid, from extant primitive fish groups.

Most advanced teleosts lack L-gulono-gamma-lactone oxidase (GULO), a key enzyme required for the biosynthesis of ascorbic acid. However, extant representatives of primitive species including sturgeon and many cartilaginous fishes, are exceptional in their ability to synthesize ascorbic acid de novo. In the present study, full-length GULO cDNAs were isolated from white sturgeon (Acipenser transmontanus) and two shark species belonging to the Triakidae (Triakis scyllium and Mustelus manazo). The open reading frames from all three species contained 440 amino acids and the deduced polypeptides had similar hydropathy profiles, predicted molecular masses and theoretical pI values. These GULO sequences exhibited high amino acid identity (67-97%) with each other, and also shared 61-71% identity with mammalian GULOs. Based on the GULO sequences obtained from these species, we developed degenerate primers for the isolation of partial GULO sequences by RT-PCR from other primitive species including another shark (Mustelus griseus, Triakidae), a spiny dogfish (Squalus acanthias, Squalidae), two ray species (Raja kenojei, Rajidae and Dasyatis akajei, Dasyatidae) and four sturgeons (Acipenser baeri, A. gueldenstaedtii, A. naccarii and A. ruthenus, Acipenseridae). Overall, sequence identities of these amplified GULO segments among primitive species were 63-99% at the nucleotide level and 67-100% at the amino acid level. Considerable numbers of amino acid residues were unique to either fish or mammals, and Acipenseriform species occupied an intermediate position, sharing several residues with either fish or mammalian GULOs. Phylogenetic analyses based on parsimony, distance and likelihood methods of both nucleotide and amino acid sequences resulted in trees that were in agreement with known taxonomy. The transcription and enzyme activity of GULO were kidney-specific when measured by biochemical assay and reverse transcription-PCR.

Multiple microarray platforms utilized for hepatic gene expression profiling of GH transgenic coho salmon with and without ration restriction.

The objectives of this study are to examine hepatic gene expression changes caused by GH transgenesis and enhanced growth. This is the first use of cDNA microarrays to study the influence of GH transgenesis on liver gene expression in a non-mammalian vertebrate, and the first such study using sexually immature animals. Three groups of coho salmon were examined: GH transgenic on full ration (T), GH transgenic on restricted ration (R), and control non-transgenic (C). Specific growth rates for weight in T were approximately eightfold higher than in C, and fourfold higher than in R. Differential gene expression in T, R, and C samples was determined using approximately 3500 and 16,000 gene microarrays, and R and C samples were compared on a different approximately 4000 gene microarray. The use of multiple microarray platforms increased the overall proportion of the hepatic transcriptome considered in these studies. Cross-platform comparisons identified genes behaving similarly between studies. For example, genes encoding a precerebellin-like protein and complement component C3 were downregulated in R relative to C (R < C) in two microarray studies, and hemoglobins alpha and beta were R > C in all three studies. Comparisons of informative gene lists within and between studies inferred causes of altered gene expression. For example, ten genes, including 78 kDa glucose-regulated protein, glycerol-3-phosphate dehydrogenase, hemoglobins alpha and beta, and a C-type lectin, were likely induced by GH transgenesis due to their presence in both T > C and R > C gene lists. Eleven genes, including hepcidin, nuclear protein p8, precerebellin-like, transketolase, and fatty acid-binding protein, were present in both T < C and R < C gene lists and were, therefore, likely suppressed by GH transgenesis. A large number of salmonid genes identified in these studies are involved in iron homeostasis, mitochondrial function, carbohydrate metabolism, cellular proliferation, and innate immunity. Pentose phosphate pathway genes phosphogluconate dehydrogenase, transaldolase, and transketolase, were dysregulated in GH transgenic samples relative to control samples. Changes in the expression of genes involved in maintaining hemoglobin levels (heme oxygenase, hemoglobins alpha and beta, Kruppel-like globin gene activator, hepcidin) in R and T fish indicate a need for additional hemoglobin in the transgenic fish, perhaps due to higher metabolic rate required for enhanced growth.

A C-terminal glycine suppresses production of pleurocidin as a fusion peptide in Escherichia coli.

The winter flounder (Pseudopleuronectes americanus) antimicrobial peptide pleurocidin was produced in Escherichia coli using a synthetic gene constructed by PCR. The gene expresses pleurocidin from pET21a fused to the C-terminus of an insoluble carrier peptide. Once expressed, the fusion peptide formed inclusion bodies in the cytoplasm that were collected, solubilized in guanidine-HCl, and chemically cleaved using hydroxylamine at a unique asparaginyl-glycyl dipeptide. This released recombinant pleurocidin (r-pleurocidin), which was purified using ultrafiltration followed by reverse phase chromatography. The r-pleurocidin peptide resolved as a single band (2.7 kDa) when analyzed by Tris-Tricine buffered SDS-PAGE, and its amino acid sequence was confirmed using tandem mass spectrometry. Extending the pleurocidin sequence with a C-terminal glycine (r-pleurocidin-G) suppressed production of the fusion peptide 15-fold. When pleurocidin was extended further to include aspartate (r-pleurocidin-GD), the same effect was observed, and when pleurocidin was extended with aspartate alone, no effect was observed. Expression of fusion peptide containing either r-pleurocidin-G or r-pleurocidin-GD with low concentrations of inductant caused E. coli to enter stationary phase prematurely, but did not affect overall growth rates. A partial production recovery of r-pleurocidin-G was achieved by inducing expression in stationary phase cells. We observed r-pleurocidin-G to have enhanced antimicrobial activity compared with r-pleurocidin, and we propose that this activity interferes with E. coli metabolism during expression. This antimicrobial effect is probably facilitated by residual solubility of the fusion peptide and by a C-terminal cap structure, which stabilizes the r-pleurocidin-G alpha-helix that is thought to be important for activity.

Identification of a transcript encoding a soluble form of toll-like receptor 5 (TLR5) in Atlantic salmon during Aeromonas salmonicida infection.

Toll-like receptors (TLRs) are involved in the innate immune response against microbial pathogens in vertebrates and insects. The extracellular region of a TLR recognizes pathogen-associated molecules, while the intracellular region initiates the signaling pathway leading to immune response. Membrane-bound TLRs have been found in most vertebrates, but few soluble forms have been reported. A novel transcript corresponding to a portion of a soluble TLR was identified in liver of infected Atlantic salmon. The complete coding sequence of this TLR was obtained and BLASTN analysis showed the highest sequence identity to a recently described full-length cDNA sequence of a soluble TLR5 from rainbow trout (GenBank Accession No.: ). The deduced protein is 40% identical to the mammalian counterpart of the leucine-rich repeat (LRR)/LRR-like motifs of TLR5. Based on the structure of human TLRs, it contains 21 LRRs with conserved LxxLxLxxNx*xx*xxxxFxxL pattern. Since TLR5 is essential for the recognition of bacterial flagellins, we hypothesize that flagellin and perhaps some other pathogen-derived factors from Aeromonas salmonicida bind to this soluble TLR through an unknown binding domain within the LRR.

Microarray studies of gene expression in fish.

The use of microarrays for the study of various aspects of fish physiology has seen a spectacular increase in recent years. From early studies with model species, such as zebrafish, to current studies with commercially important species, such as salmonids, catfish, carp, and flatfish, microarray technology has emerged as a key tool for understanding developmental processes as well as basic physiology. In addition, microarrays are being applied to the fields of ecotoxicology and nutrigenomics. A number of different platforms are now available, ranging from microarrays containing cDNA amplicons to oligomers of various sizes. High-density microarrays containing hundreds of thousands of distinct oligomers have been developed for zebrafish and catfish. As this exciting technology advances, so will our understanding of global gene expression in fish. Furthermore, lessons learned from this experimentally tractable group of organisms can also be applied to more advanced organisms such as humans.

Dietary rapeseed oil affects the expression of genes involved in hepatic lipid metabolism in Atlantic salmon (Salmo salar L.).

Supplies of marine fish oils (FO) are limited, and sustainable production in aquaculture dictates that alternatives that do not compromise fish health and product quality, such as vegetable oils, must be found. Nutrigenomics will increase our understanding of how nutrition influences metabolic pathways and homeostatic control, and may be used to measure and validate subtle changes in organ-specific, metabolic gene expression signatures. We compared 2 groups of Atlantic salmon fed diets containing 100% FO or 75% rapeseed oil (RO) for 42 wk. A small-scale cDNA microarray was constructed to screen for changes in the expression of lipid metabolism genes in the liver resulting from this partial substitution of RO for FO. Delta5 fatty acid desaturase gene expression was significantly greater in fish fed 75% RO than in fish fed the control diet; this was confirmed by quantitative real time PCR analysis. In addition, several genes, among these mitochondrial proteins, peroxisome proliferator-activated receptor gamma, as well as other transcription factors, coactivators, and signal transducers, showed significant differential regulation. This partially validated microarray may be used for further gene expression profiling using other dietary comparisons, and for further characterization of selected genes.

Structural characterization of the antimicrobial peptide pleurocidin from winter flounder.

Pleurocidin is an antimicrobial peptide that was isolated from the mucus membranes of winter flounder (Pseudopleuronectes americanus) and contributes to the initial stages of defense against bacterial infection. From NMR structural studies with the uniformly (15)N-labeled peptide, a structure of pleurocidin was determined to be in a random coil conformation in aqueous solution whereas it assumes an alpha-helical structure in TFE and in dodecylphosphocholine (DPC) micelles. From (15)N relaxation studies, the helix is a rigid structure in the membrane-mimicking environment. Strong NOESY cross-peaks from the pleurocidin to the aliphatic chain on DPC confirm that pleurocidin is contained within the DPC micelle and not associated with the surface of the micelle. From diffusion studies it was determined that each micelle contains at least two pleurocidin molecules.

Identification of genes differentially expressed in Atlantic salmon (Salmo salar) in response to infection by Aeromonas salmonicida using cDNA microarray technology.

The response of Atlantic salmon, Salmo salar, to infection by the bacterial pathogen Aeromonas salmonicida (the causative agent of furunculosis), was investigated using a cohabitation model and a custom Atlantic salmon cDNA microarray consisting of over 4000 different amplicons. Pooled samples of each of three immune-relevant tissues (spleen, head kidney and liver) were obtained from fish exposed to infected salmon for 13 days. Reverse transcription-PCR assays were used to verify the differential expression of 12 candidate genes uncovered by microarray analysis. Among the differentially expressed genes were several previously revealed by suppression subtractive hybridization and EST surveys and that are recognized to encode humoral components of the innate immune system. Other genes identified in this study were not previously associated with infection. In addition, a number of genes with no known homologs were uncovered. Determination of their specific roles during infection may lead to a better understanding of innate immunity.

Antimicrobial peptides: cooperative approaches to protection.

Reports of cationic antimicrobial peptides (CAPs) have become standard fare in research literature. But with several hundred peptides described to date, the investigator who tries to navigate the proposed models of their activity is only treated to a generous serving of incongruencies. Rather than acting in isolation as antimicrobial molecules, CAPs also may synergize with other molecules of innate immunity and modulate both innate and adaptive immune systems, thus providing a link between the various mechanisms that result in host protection.

Identification, structure and differential expression of novel pleurocidins clustered on the genome of the winter flounder, Pseudopleuronectes americanus (Walbaum).

Antimicrobial peptides form one of the first lines of defense against invading pathogens by killing the microorganisms and/or mobilizing the host innate immune system. Although over 800 antimicrobial peptides have been isolated from many different species, especially insects, few have been reported from marine fish. Sequence analysis of two genomic clones (15.6 and 12.5 kb) from the winter flounder, Pseudopleuronectes americanus (Walbaum) resulted in the identification of multiple clustered genes for novel pleurocidin-like antimicrobial peptides. Four genes and three pseudogenes (Psi) are encoded in these clusters, all of which have similar intron/exon boundaries but specify putative antimicrobial peptides differing in sequence. Pseudogenes are easily detectable but have incorrect initiator codons (ACG) and often contain a frameshift(s). Potential promoters and binding sites for transcription factors implicated in regulation of expression of immune-related genes have been identified in upstream regions by comparative genomics. Using reverse transcription-PCR assays, we have shown for the first time that each gene is expressed in a tissue-specific and developmental stage-specific manner. In addition, synthetic peptides based on the sequences of both genes and pseudogenes have been produced and tested for antimicrobial activity. These data can be used as a basis for prediction of antimicrobial peptide candidates for both human and nonhuman therapeutants from genomic sequences and will aid in understanding the evolution and transcriptional regulation of expression of these peptides.