Suppression Subtractive Hybridization cDNA Library Research Articles

Expression of Sex and Reproduction-Related Genes in Marsupenaeus japonicus

Expressed sequence tags (ESTs) were identified from reciprocal suppression subtractive hybridization cDNA libraries from Marsupenaeus japonicus (Kuruma shrimp) female and male gonads. The expression profiles of 24 of these ESTs were determined in female and male gonads and developing postlarvae by real-time quantitative reverse transcription-polymerase chain reaction. When expression was determined in gonads, six of the ESTs were expressed in ovaries only, and five of the ESTs were expressed in testes only. When expression was determined in whole individuals during postlarval development, expression of the ESTs was low and inconsistent until stage PL110 (110days since metamorphosis from mysis stage to the first postlarval stage). At PL110, seven of the ESTs were detected in females only, and seven ESTs were detected in males only. Sex-specific expression at this developmental stage indicates that these ESTs act as important gonadal development markers and may have a role in gametogenesis.

Identification of genes expressed in response to yellow head virus infection in the black tiger shrimp, Penaeus monodon, by suppression subtractive hybridization

Suppression subtractive hybridization (SSH) was employed to identify yellow head virus (YHV)-responsive genes from the hemocytes of the black tiger shrimp, Penaeus monodon. Two SSH cDNA libraries were constructed to identify viral responsive genes in the early (24I) and late (48/72I) phases of YHV infection. From 240 randomly selected clones from each library, 155 and 30 non-redundant transcripts were obtained for the early and late libraries, respectively. From these clones, 72 and 16, respectively, corresponded to known genes ( E-values < 1 × 10 −4) that could be categorized according to their putative functions. The upregulated genes identified as likely to be associated with cell defense and homeostasis were found at a high proportion in the 24I SSH library, but not in 48/72I SSH library implying that these immune molecules participate in viral defense immunity in the early phase of YHV infection whereas their expressions were suppressed in the late phase of infection. Novel YHV-responsive genes were uncovered from these SSH libraries including caspases, histidine triad nucleotide-binding protein 2, Rab11, β-integrin, tetraspanin, prostaglandin E synthase, transglutaminase, Kazal-type serine proteinase inhibitor and antimicrobial peptides. Among these YHV-responsive genes, several have been previously reported to participate in defense against white-spot syndrome virus (WSSV) implying that YHV infection in shrimp induces similar host immune responses as observed during WSSV infection. The expression of four apparently upregulated immune-related genes identified from the two SSH libraries, anti-lipopolysaccharide factor isoform 6 (ALF Pm6), crustin isoform 1 (crustin Pm1), transglutaminase and Kazal-type serine proteinase inhibitor isoform 2 (SPI Pm2), was evaluated by real-time RT-PCR to reveal differential expression in response to YHV infection at 6, 24, 48 and 72 h post-infection. The results confirmed their differential expression and upregulation, and thus verified the success of the SSHs and the likely involvement of these genes in shrimp antiviral mechanisms.

Comparison of Gene Expression between Cumulus Oocyte Complexes and Naked Oocytes by Suppression Subtractive Hybridization in Swine

In the antral follicle phase, several layers of cumulus cells surround the oocyte and play an important support and regulation role in oocyte development and maturation via intercellular communications and interactions between oocytes and cumulus cells. However, information on stage specific gene expression in swine during the phase is not well understood. To investigate the function of cumulus cells during in vitro maturation of porcine oocytes and gene expression, suppression subtractive hybridization (SSH) was performed to screen genes that were differentially expressed between cumulus-oocyte complexes (COCs) and naked oocytes (NOs). Utilizing mRNAs from in vitro maturation oocytes, a SSH cDNA library from COCs as the tester and NOs as the driver was constructed. The SSH cDNA library was then screened using dot blot analysis. Results showed that a total of 70 clones randomly selected from the library were differentially expressed. Among these, 41 exhibited high homology to known genes and 11 were novel expressed sequences tags (ESTs). Four differentially expressed genes, including bfgf, sprouty 2, egr and btc, were further studied by real time quantitative PCR; results confirmed an increased expression of respective mRNA in COCs compared with NOs, which suggests that these factors may play an important role in oocyte development and maturation.

Cloning and expression analysis of a ribosomal protein S7 in wheat under the stress of Puccinia triticina

A gene was isolated from the suppression subtractive hybridization (SSH) cDNA library of wheat by reverse northern blotting. Full length cDNA was obtained by RACE technique, and the result showed that all sequences consisted of 471 bp and encoded 157 amino acid. Subsequently, the gene expression was enhanced according to quantity real-time PCR (qRT-PCR) in response to leaf rust stress as the time over. All the results above suggested that the gene might play important role in wheat hypersensitive response (HR) to leaf rust.

Functional analysis ofTaDi19A, a salt-responsive gene in wheat

A salinity stress upregulated expressed sequence tag (EST) was selected from a suppression subtractive hybridization cDNA library, constructed from the salinity-tolerant wheat cultivar Shanrong No. 3. Sequence analysis showed that the corresponding gene (named TaDi19A) belonged to the Di19 family. TaDi19A was constitutively expressed in both the root and leaf of wheat seedlings grown under non-stressed conditions, but was substantially up-regulated by the imposition of stress (salinity, osmotic stress and cold), or the supply of stress-related hormones [abscisic acid (ABA) and ethylene]. The heterologous over-expression of TaDi19A in Arabidopsis thaliana increased the plants' sensitivity to salinity stress, ABA and mannitol during the germination stage. Root elongation in these transgenic lines showed a reduced tolerance to salinity stress and a reduced sensitivity to ethophon. The expression of the ABA signal pathway genes ABI1, RAB18, ERD15 and ABF3, and SOS2 (SOS pathway) was altered in the transgenic lines. TaDi19A plays a role in the plant's response to abiotic stress, and some possible mechanisms of its action are proposed.

Identification of genes differentially expressed in hemocytes of Scylla paramamosain in response to lipopolysaccharide

Although the crab Scylla paramamosain has been cultured in China for a long time, little knowledge is available on how crabs respond to infection by bacteria. A forward suppression subtractive hybridization (SSH) cDNA library was constructed from their hemocytes and the up-regulated genes were identified in order to isolate differentially expressed genes in S. paramamosain in response to bacterial lipopolysaccharide (LPS). A total of 721 clones on the middle scale in the SSH library were sequenced. Among these genes, 271 potentially functional genes were recognized based on the BLAST searches in NCBI and were categorized into seven groups in association with different biological processes using AmiGO against the Gene Ontology database. Of the 271 genes, 269 translatable DNA sequences were predicted to be proteins, and the putative amino acid sequences were searched for conserved domains and proteins using the CD-Search service and BLASTp. Among 271 genes, 179 (66.1%) were annotated to be involved in different biological processes, while 92 genes (33.9%) were classified as an unknown-function gene group. It was noted that only 18 of the 271 genes (6.6%) had previously been reported in other crustaceans and most of the screened genes showed less similarity to known sequences based on BLASTn results, suggesting that 253 genes were found for the first time in S. paramamosain. Furthermore, two up-regulated genes screened from the SSH library were selected for full-length cDNA sequence cloning and in vivo expression study, including Sp-superoxide dismutase ( Sp-Cu-ZnSOD) gene and Sp-serpin gene. The differential expression pattern of the two genes during the time course of LPS challenge was analyzed using real-time PCR. We found that both genes were significantly expressed in LPS-challenged crabs in comparison with control. Taken together, the study primarily provides the data of the up-regulated genes associated with different biological processes in S. paramamosain in response to LPS, by which the interesting genes or proteins potentially involved in the innate immune defense of S. paramamosain will be investigated in future.

Transcript responses in leaves of ozone-treated beech saplings seasons at an outdoor free air model fumigation site over two growing seasons

The still most abundant air pollutant, tropospheric ozone, leads to severe oxidative stress in plants. To investigate the ozone responsiveness of trees, we carried out a microarray analysis of beech saplings, grown around a free air lysimeter station in the years 2005 and 2006. The microarrays revealed ozone-responsive expressed sequence tags (ESTs), derived from a suppression subtractive hybridisation cDNA library. Chronic ozone exposure effects were investigated at different time points throughout the whole vegetation period in 2005 and 2006. Significantly regulated ESTs were calculated with R-packages. Different transcript patterns in both years could be related to weather conditions and consequential a different ozone uptake. Fewer transcriptome changes were found in 2005 compared to 2006. Induction of ESTs involved in cell structure, related to stress response and cell walls, signal transduction, as well as disease and defence in July and August indicated ozone-related stress responses in both years. An early response to ozone on primary metabolism, or, more precisely, on plant growth and photosynthesis, was observed in July and August particularly in the year 2006. This study demonstrated a clear transcriptional ozone response of juvenile beech, the most important deciduous tree in Central Europe, under free air conditions.

Disruption of the Salmon Reproductive Endocrine Axis Through Prolonged Nutritional Stress: Changes in Circulating Hormone Levels and Identification of Differentially Expressed Ovarian Genes.

Environmental stressors (e.g., reduced food availability, hypoxia, endocrine disrupting chemicals) are known to disrupt endocrine function of fish, potentially delaying the age of puberty and affecting gamete quality. Assessing the reproductive health of fish in early stages of gametogenesis using histological and hormonal indices provides useful information, but has limited prognostic value for future reproductive potential. Our long-term aim is to develop tools for monitoring environmental impacts on ovarian growth in fish using large-scale ovarian gene expression analyses. However, little is known about the regulation of gonadal gene expression during previtellogenic oocyte growth in either healthy growing fish or fish exposed to environmental conditions that have been shown to disrupt the reproductive endocrine system and inhibit gonadal growth. The goal of this study was to identify genes that are specifically up- or down-regulated in the previtellogenic ovary of coho salmon when ovarian growth is impaired by prolonged fasting. Two year-old fish were individually tagged and fasted over a 17-week period, which resulted in less than 20% loss of body weight. Prolonged fasting caused regression of the ovaries and disrupted the reproductive endocrine axis, as fasted salmon showed significantly lower gonad weights, oocyte diameters, and plasma estradiol-17β (E2) levels relative to fed animals by week 14. Further, a higher proportion of atretic oocytes was observed in ovaries of fasting fish at week 14 and numbers increased by week 17. Reciprocal suppression subtractive hybridization (SSH) cDNA libraries using ovaries from fed and fasted animals at week 14 were generated. A total of 480 clones were sequenced per library and their putative identity determined by comparing sequences to the NCBI and fish EST databases. The library representing genes up-regulated in the fed ovary included steroidogenesis related genes, such as steroidogenic acute regulatory protein (star), 3β-hydroxysteroid dehydrogenase (hsd3b), P450-aromatase (cyp19a1a), and TGF-β superfamily members, such as anti-Mullerian hormone (amh) and inhibin (inha). The library representing genes up-regulated in the fasted ovary included genes that have been associated with apoptosis, such as programmed cell death protein 4 (pdcd4), lipopolysaccharide-induced TNF factor (litaf), cullin 3 (cul3), and kruppel-like factor 6 (klf6). The genes described above were examined with quantitative PCR and results confirmed that the SSH libraries were enriched in transcripts differentially expressed between ovaries of fed and fasted salmon. This study has revealed ovarian genes that are important to normal ovarian development and those that are affected by nutritional stress and may relate to apoptosis/atresia within the ovary. Ultimately, this and subsequent studies should provide new tools that will enable molecular based assessment of fish reproductive health. This research was funded by Washington Sea Grant project number R/B-49. (poster)

Identification of the molecules involved in zebra mussel (Dreissena polymorpha) hemocytes host defense

The invasion of North American waterbodies by the zebra mussel (Dreissena polymorpha) has caused ecological catastrophies in North America. Unfortunately, little is known about this nuisance mollusk and its host defense mechanisms. In this study, 32 expressed sequence tags (ESTs) associated with hemocyte stimulation were obtained from a suppression subtractive hybridization (SSH) cDNA library. This SSH-cDNA library was produced by using a cDNA library of naïve hemocytes as the driver and a cDNA library of hemocytes stimulated with a mixture of microbial antigens namely, lipopolysacchride (LPS), peptidoglycan (PGN), and zymosan (ZYM), as the tester. The driver cDNA was subtracted from the tester to increase the relative abundance of the cDNAs that were induced by stimulations. The putative function of 27 ESTs were obtained by using the homologue searching program BLASTx and BLASTn. Four ESTs encoding the protein product homologous to matrilin (Matrn, AM503947), heat shock protein 70 (HSP70, EU835391), seryl-tRNA synthetase (STS, AM503950), and glycine-rich protein (GRP, AM502279) were selected for a subsequent study using quantitative PCR (qPCR) assays with the RNA extracted from hemocytes stimulated with LPS alone. The results of quantitative PCR with stimulated hemocyte RNA demonstrated that the four candidate genes were upregulated by LPS stimulation. The expression levels of both HSP70 and Matrn genes between naïve and 1-h stimulated hemocyte samples are the most significant with 2.78 and 2.20 fold increases, respectively. The significant changes of GRP and STS genes were observed after 2-h stimulation. The phylogenetic analysis of HSP70 molecule indicated that this protein is phylogenetically close to the HSP70 identified from other mollusks. This study shed light on hemocyte-mediated host defense mechanisms of D. polymorpha.

Differentially expressed transcripts from cucumber ( Cucumis sativus L.) root upon inoculation with Fusarium oxysporum f. sp. cucumerinum Owen

Fusarium wilt of cucumber is a serious vascular disease worldwide. The molecular interaction between cucumber ( Cucumis sativus L.) and Fusarium oxysporum was investigated by construction and characterization of a suppression subtractive hybridization cDNA library, which was constructed from F. oxysporum-inoculated cucumber. To screen the differentially expressed genes, dot blotting was used with cDNA probes prepared from mRNAs of control and F. oxysporum-inoculated samples. The elevated expression levels and overall profiles over time of eight genes were conformed by semi-quantitative RT-PCR analysis. Acquired positive clones were subjected to sequence and homology search analysis. In total, 153 ESTs were acquired after screening. Among these 153 ESTs, 80 ESTs showed significant sequence similarity to known genes, 51 had unknown functions, 22 clones did not match any known sequences. Most of the ESTs with known functions were involved in defense response, metabolism and transcriptional regulation. So upon F. oxysporum inoculation, cucumber could express defense-related genes as well as enhance its metabolic activities. The most interesting finding was that several genes were related to ABA, suggesting ABA singling may play a positive role in defense mechanism in cucumber to F. oxysporum. Our study provides an overview of gene expression in F. oxysporum-inoculated cucumber, as well as useful information of the defense mechanism in plant to necrotrophic fungi pathogens.

Identification and analysis of differentially expressed genes in immune tissues of Atlantic cod stimulated with formalin-killed, atypicalAeromonas salmonicida

Physiological changes, elicited in animal immune tissues by exposure to pathogens, may be studied using functional genomics approaches. We created and characterized reciprocal suppression subtractive hybridization (SSH) cDNA libraries to identify differentially expressed genes in spleen and head kidney tissues of Atlantic cod (Gadus morhua) challenged with intraperitoneal injections of formalin-killed, atypical Aeromonas salmonicida. Of 4,154 ESTs from four cDNA libraries, 10 genes with immune-relevant functional annotations were selected for QPCR studies using individual fish templates to assess biological variability. Genes confirmed by QPCR as upregulated by A. salmonicida included interleukin-1 beta, interleukin-8, a small inducible cytokine, interferon regulatory factor 1 (IRF1), ferritin heavy subunit, cathelicidin, and hepcidin. This study is the first large-scale discovery of bacteria-responsive genes in cod and the first to demonstrate upregulation of IRF1 in fish immune tissues as a result of bacterial antigen stimulation. Given the importance of IRF1 in vertebrate immune responses to viral and bacterial pathogens, the full-length cDNA sequence of Atlantic cod IRF1 was obtained and compared with putative orthologous sequences from other organisms. Functional annotations of assembled SSH library ESTs showed that bacterial antigen stimulation caused changes in many biological processes including chemotaxis, regulation of apoptosis, antimicrobial peptide production, and iron homeostasis. Moreover, differences in spleen and head kidney gene expression responses to the bacterial antigens pointed to a potential role for the cod spleen in blood-borne pathogen clearance. Our data show that Atlantic cod immune tissue responses to bacterial antigens are similar to those seen in other fish species and higher vertebrates.

Development of a cDNA microarray of zebra mussel ( Dreissena polymorpha) foot and its use in understanding the early stage of underwater adhesion

The underwater adhesion of the zebra mussel ( Dreissena polymorpha) to substrates is a complex process that is controlled by a delicate apparatus, the byssus. As a critical activity of the byssus glands embedded in the zebra mussel feet, byssogenesis is highly active to produce numerous byssal threads from the settled juvenile stage through the adult stage in its life cycle. This lifelong activity helps the zebra mussel to firmly attach to substrata underwater, thereby causing severe economic and ecologic impacts. In an attempt to better understand the zebra mussel's byssus activity, a cDNA microarray (ZMB) including 716 genes, generated from a Suppression Subtractive Hybridization (SSH) cDNA library, was printed and used for the comparison of gene expression during zebra mussel adhesion and non-adhesion. To better understand the byssogenesis mechanism, RNA samples from the zebra mussel feet with byssogenesis and without byssogenesis were used in a two-color hybridization to reveal the gene differential expression in the two states. Based on the P values ( P < 0.05), Fifty-two ESTs were found as differentially expressed genes and were divided into two groups, upregulated and downregulated groups according to there logFC values. With the false discovery rate (FDR) adjustment, seven were identified from the upregulated group and nine from the downregulated group. Phylogenetic analysis indicated that the four excretory gland peptide-like protein (EGP) encoding genes in upregulated group are structurally different than the two in the downregulated list. The amino acid composition analysis on the proteins, which were encoded by the up- or downregulated ESTs without homologues (NH) suggested that seven of the NH proteins are biochemically similar to the novel foot proteins from other mussels. The quantitative reverse transcription PCR (QRT-PCR) proved the uniqueness of the templates in the array, and also confirmed the differentially expressed genes identified by microarray experiment. Our findings demonstrated that the zebra mussel byssus cDNA microarray is an efficient tool for the studies of differential gene expression in different byssogenesis states, thereby revealing important details of the underwater adhesion.

Genes Induced in Response to Mercury-Ion-Exposure in Heavy Metal HyperaccumulatorSesbania drummondii

Sesbania drummondii plants have been recognized as a potential mercury (Hg) hyperaccumulator. To identify genes modulated by Hg, two suppressive subtraction hybridization (SSH) cDNA libraries (forward and reverse) were constructed. A total of 348 differentially expressed clones were isolated and 95 of them were identified as Hg responsive. Reverse Northern results showed that 31 clones from forward library were down-regulated and 64 clones from reverse library were up-regulated in Hg-treated plants. Sixty-seven of them showed high homology to genes with known or putative function, and 28 were uncharacterized genes. Two full-length cDNAs coding for a putative metallothionein type 2 protein (SdMT2) and an auxin responsive protein (SdARP) were isolated and characterized. The expression levels of SdMT2 and SdARP increased 3- and 5-fold, respectively. Results suggest that up-regulated expression of SdARP may contribute to the survival of Sesbania plants under mercury stress, whereas SdMT2 is likely to be involved in alleviation of Hg toxicity. The possible correlation between gene expression and heavy metal tolerance of Sesbania plants is discussed.

Suppression subtractive hybridization (SSH) for isolation and characterization of genes related to testicular development in the giant tiger shrimp Penaeus monodon

Suppression subtractive hybridization (SSH) cDNA libraries of the giant tiger shrimp, Penaeus monodon, were constructed. In total, 178 and 187 clones from the forward and reverse SSH libraries, respectively, of P. monodon were unidirectionally sequenced. From these, 37.1% and 53.5% Expressed Sequence Tags (ESTs) significantly matched known genes (E-value < 1e-04). Three isoforms of P. monodon progestin membrane receptor component 1: PM-PGMRC1-s (1980 bp), PM-PGMRC1-m (2848 bp), and PM-PGMRC1-l (2971 bp), with an identical ORF of 573 bp corresponding to a deduced polypeptide of 190 amino acids, were successfully identified by RACE-PCR. Interestingly, PMPGMRC1 showed a greater expression level in testes of juvenile than broodstock P. monodon (P < 0.05). Dopamine administration (10(-6) mol/shrimp) resulted in up-regulation of PMPGMRC1 in testes of juveniles at 3 hrs post treatment (P < 0.05), but had no effect on PM-Dmc1 (P > 0.05).

Identification of new up-regulated genes under drought stress in soybean nodules

Legumes/rhizobium biological N 2 fixation (BNF) is dramatically affected under abiotic stress such as drought, salt, cold and heavy metal stresses. Nodule response to drought stress at the molecular level was analysed using soybean ( Glycine max) and Bradyrhizobium japonicum as a model, since this symbiotic partnership is extremely sensitive to this stress. To gain insight into molecular mechanisms involved in drought-induced BNF inhibition, we have constructed a SSH (Suppression Subtractive Hybridisation) cDNA library from nodular tissue of plants irrigated at field capacity or plants water deprived for 5 days. Sequence analysis of the first set of 128 non redundant ESTs using protein databases and the Blastx program indicated that 70% of ESTs could be classified into putative known functions. Using reverse northern hybridization, 56 ESTs were validated as up-regulated genes in response to drought. Interestingly, only a few of them had been previously described as involved in plant response to drought, therefore most of the ESTs could be considered as new markers of drought stress. Here we discuss the potential role of some of these up-regulated genes in response to drought. Our analysis focused on two genes, encoding respectively a ferritin and a metallothionein, which are known to be involved in homeostasis and detoxification of metals and in response to oxidative stress. Their spatiotemporal expression patterns showed a high accumulation of transcripts restricted to infected cells of nodules in response to drought.

Distal intestinal gene expression in Atlantic salmon (Salmo salarL.) fed genetically modified maize

In the current experiment, RNA was isolated from the distal intestine (DI) of Atlantic salmon-fed fishmeal-based diets containing either genetically modified (GM) maize (Bt maize, Mon810®, Monsanto Company, St. Louis, Missouri, USA) or its conventional near-isogenic parental line (non-GM) for 82 days, both at 300 g kg−1 inclusion. From a suppression subtractive hybridization (SSH) cDNA library, 192 clones with similarity to both known and novel Atlantic salmon sequences were identified. Real-time PCR was used to study the differential expression of 10 clones between the dietary groups. Expression of a clone showing high protein similarity to proton-dependent high-affinity oligopeptide transporter was significantly upregulated in fish-fed GM maize compared with fish-fed non-GM maize. No significant differences in expression were observed for the nine other clones showing similarity to the following proteins: heat shock protein 90B, procathepsin B, interferon gamma-inducible protein 30, ferritin heavy subunit, serum lectin isoform/C-type mannose-binding lectin, fatty acid-binding protein/gastrotropin, ATP synthase [H+ transporting, mitochondrial F0 complex, subunit c (ATPSYNT)], sonic hedgehog and translationally controlled tumour protein. In conclusion, only minor differences in DI transcriptional gene expression was observed between fish fed the GM and non-GM maize diets.

Dehydrins expression related to timing of bud burst in Norway spruce

Cold deacclimation and preparation to flushing likely requires rehydration of meristems. Therefore, water stress related genes, such as dehydrins (DHN), might play an important role in providing protection during winter dormancy, deacclimation and bud burst timing processes. Here we report the sequence analysis of several Norway spruce DHN identified in late and early flushing suppressive subtraction hybridization cDNA libraries and in our Norway spruce EST database. We obtained 15 cDNAs, representing eight genes from three distinct types of DHN, and studied differential expression of these genes before and during bud burst in spring, using qRT-PCR. We found the visible reduction in transcript level of most DHN towards the bud burst, supported by a significant down-regulation of the DHN in needles during experimental induction of bud burst applied at three time points during autumn in Norway spruce grafts. For most of the DHN transcripts, their expression levels in late-flushing spruces were significantly higher than in the early flushing ones at the same calendar dates but were remarkably similar at the same bud developmental stage. From our results we may conclude that the difference between the early and the late families is in timing of the molecular processes leading to bud burst due to differences in their response to the increasing temperature in the spring. They are induced much earlier in the early flushing families.

Molecular characterisation of a goose-type lysozyme gene in Atlantic cod ( Gadus morhua L.)

Lysozymes are antibacterial enzymes important in the innate immune defense of several animal phyla. An Atlantic cod goose-type (g-type) lysozyme EST was identified in a suppression subtractive hybridisation (SSH) cDNA library and the full-length cDNA ( codg1) was obtained by RACE-PCR. The lysozyme gene is organised in five exons and four introns similar to g-type lysozyme genes in other fish species. Two different cod lysozyme transcripts, named codg1 and codg2, seem to be produced by the use of alternative transcription start sites (TSS) in the lysozyme gene. The alternative TSS cause a different exon I usage where exon Ia transcripts possess a putative signal peptide ( codg1) while exon Ib transcripts ( codg2) lack this feature. Lysozyme without the signal peptide was produced recombinantly in Escherichia coli and displayed muramidase activity against Micrococcus luteus cells at an unusually low pH. Gene expression analysis of codg1 and codg2 showed that both were expressed in several tissues with highest expression in the head kidney, peritoneum and spleen. Codg1 and codg2 were differentially expressed in some tissues. In the non-immunised control group, codg2 was expressed significantly higher in the head kidney compared to codg1, while an opposite expression profile was observed in the gills. Compared to non-immunised fish, a significant up-regulation of codg2 transcripts was observed in the peritoneum and gills after injection of formalin inactivated Listonella anguillarum indicating a role for g-type lysozyme in the innate defense system of cod.

Putative identification of expressed genes associated with attachment of the zebra mussel (Dreissena polymorpha)

Because of its aggressive growth and firm attachment to substrata, the zebra mussel (Dreissena polymorpha) has caused severe economic and ecological problems since its invasion into North America. The nature and details of attachment of this nuisance mollusc remains largely unexplored. Byssus, a special glandular apparatus located at the root of the foot of the mussel produces threads and plates through which firm attachment of the mollusc to underwater objects takes place. In an attempt to better understand the adhesion mechanism of the zebra mussel, the suppression subtractive hybridization (SSH) assay was employed to produce a cDNA library with genes unique to the foot of the mussel. Analysis of the SSH cDNA library revealed the presence of 750 new expressed sequence tags (ESTs) including 304 contigs and 446 singlets. Using BLAST search, 365 zebra mussel ESTs showed homology to other gene sequences with putative functions. The putative functions of the homologues included proteins involved in byssal thread formation in zebra and blue mussels, exocrine gland secretion, host defence, and house keeping. The generated data provide, for the first time, some useful insights into the foot structure of the zebra mussel and its underwater adhesion.

Functional genomic analysis of the response of Atlantic cod ( Gadus morhua) spleen to the viral mimic polyriboinosinic polyribocytidylic acid (pIC)

In order to improve our understanding of how Atlantic cod (Gadus morhua) respond to viruses, we characterized immune-related gene expression in spleen tissues following stimulation with a synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid (pIC). We used reciprocal suppression subtractive hybridization (SSH) cDNA libraries and quantitative RTPCR (QPCR) to identify and quantify pIC-responsive transcripts. A total of 3874 expressed sequence tags (ESTs) were generated from SSH libraries enriched for genes responsive to pIC. Thirteen immune-relevant genes from the libraries were subjected to QPCR. Genes confirmed as up-regulated by pIC included interferon stimulated gene 15, a small inducible cytokine, interferon regulatory factors (1, 7, and 10), MHC class I, viperin, and ATP-dependent helicase LGP2. Alpha-1-microglobulin (bikunin) was down-regulated, suggesting that pIC may suppress the acute phase response. Since the SSH libraries built for this study identified genes involved in the antiviral response, they are important resources for studying the responses of Atlantic cod to viruses. Evidence is provided for the existence of a RIG-I-like RNA helicase viral recognition pathway in Atlantic cod. Taken together, our data show that Atlantic cod can recognize double-stranded RNA and mount a rapid and potent interferon pathway response that is similar to that observed in other fish species and higher vertebrates.