Subtractive Hybridization Method Research Articles

Differential gene expression in the brain of Sebastiscus marmoratus in response to exposure to polychlorinated biphenyls (PCBs)

Effects of exposure to polychlorinated biphenyls (PCBs) on Sebastiscus marmoratus were investigated using a suppression subtractive hybridization method. A total of 108 gene sequences were identified as having the potential for being differentially expressed, and 45 could be identified with homologous database sequences. Functions with which they were associated included long-term potentiation and neurotransmitter release, neuroendocrine, mitosis and cell proliferation, energy-related metabolism, general metabolism, signal protein, hemopoiesis system, immune system, and structure. The expression of 17 of these genes was analyzed in the brain using real time fluorescent quantitative PCR. The present study provided a basis for studying the response of fish to PCB exposure and allowed the characterization of new potential neurotoxicol biomarkers of PCB contamination in seawater.

Squalene epoxidase, located on chromosome 8q24.1, is upregulated in 8q+ breast cancer and indicates poor clinical outcome in stage I and II disease

Gains of chromosomes 7p and 8q are associated with poor prognosis among oestrogen receptor-positive (ER+) stage I/II breast cancer. To identify transcriptional changes associated with this breast cancer subtype, we applied suppression subtractive hybridisation method to analyse differentially expressed genes among six breast tumours with and without chromosomal 7p and 8q gains. Identified mRNAs were validated by real-time RT–PCR in tissue samples obtained from 186 patients with stage I/II breast cancer. Advanced statistical methods were applied to identify associations of mRNA expression with distant metastasis-free survival (DMFS). mRNA expression of the key enzyme of cholesterol biosynthesis, squalene epoxidase (SQLE, chromosomal location 8q24.1), was associated with ER+ 7p+/8q+ breast cancer. Distant metastasis-free survival in stage I/II breast cancer cases was significantly inversely related to SQLE mRNA in multivariate Cox analysis (P<0.001) in two independent patient cohorts of 160 patients each. The clinically favourable group associated with a low SQLE mRNA expression could be further divided by mRNA expression levels of the oestrogen-regulated zinc transporter LIV-1. The data strongly support that SQLE mRNA expression might indicate high-risk ER+ stage I/II breast cancers. Further studies on tumour tissue from standardised treated patients, for example with tamoxifen, may validate the role of SQLE as a novel diagnostic parameter for ER+ early stage breast cancers.

Up-regulation of kinesin light-chain 1 gene expression by acetyl- l-carnitine: Therapeutic possibility in Alzheimer's disease

We investigated the effects of acetyl- l-carnitine on gene expression by means of the suppression subtractive hybridization method. The approach gives the generation of subtracted cDNA libraries and the subsequent identification of differentially expressed transcripts after treatment of rats with acetyl- l-carnitine for 21 days. We observed that acetyl- l-carnitine increases the light-chain subunit of kinesin-1 gene expression. Recent evidences reported a link between kinesin-1 light-chain and Alzheimer's disease. Pathological hallmarks of Alzheimer's disease are potentially linked to alterations of the axonal compartments. Amyloid-beta peptide is a principal component of senile plaques and is considered to be central in the pathogenesis of the disease. The fast anterograde axonal transport of amyloid-beta peptide is mediated by direct binding to the light-chain subunit of kinesin-1. In this scenario, our results are of relevant importance for possible therapeutic intervention, suggesting a pathway for the treatment of Alzheimer's disease.

Identification of a putative tetrasporophyte-specific gene in Gracilaria lemaneiformis (Gracilariales, Rhodophyte)

A putative tetrasporophyte-specific gene, designated as SSH466 (GenBank accession No. DQ019223), was one of the genes identified in this work using suppression subtractive hybridization (SSH) method in Gracilaria lemaneiformis. The full length of the gene was obtained using SMART RACE strategy. Sequence analysis revealed that the gene had 1 019 nucleotides, including an open reading frame of 498 nucleotides encoding 166 amino acid residues, 158 nucleotides of 5′ untranslated region and 363 nucleotides of 3′ non-coding region. Protein motif and secondary structure prediction showed that there existed a transmembrane domain with a unique β-sheet. Thus, SSH466 protein might be a cross-membrane protein. Sequence homology search in the public GenBank databases did not reveal any significant match with SSH466. Virtual Northern blot analysis confirmed that it was a tetrasporophyte-specific gene.

Differentially expressed genes in resistant and susceptible Bombyx mori strains infected with a densonucleosis virus

We investigated variations in the gene expression of Bombyx mori following infection with a densonucleosis virus (BmDNV-Z). Two B. mori near-isogenic lines, Jingsong and Jingsong.nsd-Z.NIL, which are highly susceptible and completely resistant to BmDNV-Z, respectively, were used in this study. The infection profiles of BmDNV-Z in the midguts of the B. mori Jingsong and Jingsong.nsd-Z.NIL larvae revealed that the virus invaded the midguts of both of these strains. However, its proliferation was notably inhibited in the midgut of the resistant strain. By using the suppression subtractive hybridization method, three cDNA libraries were constructed to compare BmDNV-Z responsive gene expression between the two silkworm lines. In total, 151 differentially expressed genes were obtained. Real-time qPCR analysis confirmed that 11 genes were significantly up-regulated in the midgut of the Jingsong.nsd-Z.NIL strain following BmDNV-Z infection. Our results imply that these up-regulated genes might be involved in B. mori immune responses against BmDNV infection.

Construction of cDNA Library from Fertility Related Genes of Male Sterile Wheat with Aegilops kotschyi Cytoplasm by SSH

Male sterility with Aegilops kotschyi cytoplasm has a great potential in hybrid wheat ( Triticum aestivum L.) breeding for its stable sterility and broad-spectrum of restoring gene resources. To further study the genetic mechanism of male sterility with Ae. kotschyi cytoplasm, a male sterile line ms(Kots)-90-110(A) and its near isogenic line BC 4F 1 (fertility restored by rk5451) were used to construct sterility and fertility cDNA libraries, using the suppression subtractive hybridization (SSH) method. Wheat anthers with pollen mother cells at the dikaryophase were sampled for RNA extraction via microscopic examination. Both cDNA libraries were good in quality with high efficiency of SSH. A total of 120 positive clones were randomly selected and sequenced from the 2 libraries, and 100 high quality sequences were obtained. According to BLAST screening and functional annotation, energy-related genes were identified with high frequencies in the fertility cDNA library, whereas, MADS-box transcriptional factor TaAGL7, ubiquitin-conjugating enzyme, and adenosine diphosphate glucose pyrophosphatase were detected in the sterility cDNA library. The MADS-box transcription factor TaAGL7 played an important role in regulating flower development. The immature spike ubiquitin-conjugating enzyme would probably relate to apoptosis. The expression of adenosine diphosphate glucose pyrophosphatase directly affected starch synthase and the normal supply of energy. The genes detected in the present study are probably important genes associated with the fertility in wheat with Ae. kotschyi cytoplasm.

Isolation and characterization of LHY homolog gene expressed in flowering tissues of Tectona grandis (teak)

Floral initiation of teak through molecular biology approach is being studied for better understanding of teak flower development. Through PCR subtractive hybridization method, LHY homolog gene has been isolated from teak flowering tissues. The full-length cDNA of the gene was 2948 base pair (bp) and potentially encoded for 768 amino acids. It was named Tectona grandis LHY (Tg-LHY), as the gene was similar to the LHY gene of some species. Amino acid sequence alignment revealed that Tg-LHY was similar to LHY of Castanea sativa, LHY of Phaseolus vulgaris and LHY of Arabidopsis thaliana. The highly conserved region found in Tg-LHY was the MYB protein, which is the DNA-binding protein responsible in negative feedback loop reaction of central oscillator in plant circadian clock system. The level of gene expression was found to be high four hours after dawn in flowering shoots and flower. This paper reported the isolation and characterization of the gene.

Isolation and characterization of 12 dinucleotide microsatellite loci in the anadromous fish Osmerus eperlanus (L.)

A subtractive hybridization method was used to isolate 12 dinucleotide microsatellite loci for the anadromous European smelt, Osmerus eperlanus (L.). Three to 17 (mean 8.08) alleles per locus were identified in the two populations screened, and observed heterozygosity ranged from 0.067 to 0.933. Loci in both populations showed significant deviations from Hardy-Weinberg expected frequencies. These 12 loci provide a good basis for investigation of O. eperlanus population structure.

Characterization of the BPI-like gene from a subtracted cDNA library of large yellow croaker ( Pseudosciaena crocea) and induced expression by formalin-inactivated Vibrio alginolyticus and Nocardia seriolae vaccine challenges

One expressed sequence tag (EST 64LF004 clone), which is from the subtracted cDNA library of the head kidney of large yellow croaker ( Pseudosciaena crocea) stimulated with peptidoglycan (PG) by suppression subtractive hybridization (SSH) method, was cloned using RACE–PCR. The full length cDNA, which possesses typical structural features of a signal peptide, a conserved LPS binding domain and two bactericidal permeability-increasing (BPI) motifs as in higher vertebrates, was identified as a novel homologue, namely of the large yellow croaker BPI-like molecule (Pc-BPI-L). Phylogenetic analysis showed this Pc-BPI-L of large yellow croaker as the most ancestral branch in bony fish clade. The recombinant Pc-BPI-L protein expressed in the Tn-5B1-4 insect cells was successfully produced and confirmed to have the predicted size of 52 kDa by Western blot analysis. At the message level, Pc-BPI-L mRNA was ubiquitously expressed in all tissues examined. Following formalin-inactivated Vibrio alginolyticus and Nocardia seriolae treatment, Pc-BPI-L message was differentially up-regulated in primary immune organs. These results indicate that Pc-BPI-L might be involved in the immune response to bacterial infection.

Differential gene expression in oyster exposed to sewage

In order to investigate the influence of domestic sewage on the gene expression of Pacific oysters Crassostrea gigas, suppression subtractive hybridization (SSH) method was employed. Oysters were sampled at a farming area and, after 10 days of acclimation in the laboratory, were exposed to untreated domestic sewage diluted 33% for 48 h. Gills of male oysters were excised for total RNA extraction. mRNA was purified and the differential gene expression was analyzed by SSH. We obtained 61 cDNA sequences but only 15 were identified, which includes fatty acid binding protein, multidrug resistance protein, omega glutathione S-transferase, cytochrome P450 isoform CYP356A1, among others. The identified genes are associated with different metabolic functions like biotransformation, membrane transport, aerobic metabolism and translational machinery, evidencing the potential toxic effects elicited by these effluents.

Identification and characterization of differentially expressed genes involved in pharmacological activities of roots of Panax notoginseng during plant growth

Panax notoginseng is a highly valued Chinese medicinal herb. To understand the molecular mechanism of the much higher pharmacological activities of roots of 3-year-old plants over 1-year-old ones,two cDNA libraries were constructed using the suppression subtractive hybridization (SSH) method. Positive cDNA clones from each of the two libraries were randomly selected for dot-blotting analysis. A total of 110 genes were highly expressed in 3-year-old roots and 80 genes in 1-year-old roots. Of these, 87 cDNA fragments were sequenced, assembled, and compared with sequences in GenBank, and 81 individual cDNAs were identified. These cDNAs were the first expressed sequence tags of P. notoginseng in GenBank. The result of reverse transcription PCR analysis of six genes was consistent with that of the dot-blot analysis. The global gene expression profile showed that there were significant differences between 1- and 3-year-old roots of P. notoginseng plants. Some important structural and regulatory genes which may be involved in isoprenoid biosynthesis were found to be over-expressed in 3-year-old roots, such as genes encoding 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphat-synthase (IspG-protein), multi-copper oxidase type I family protein, NADH flavin oxidoreductase, lipase and aconitase.

Transcriptomic identification of candidate genes involved in sunflower responses to chilling and salt stresses based on cDNA microarray analysis

BackgroundConsidering that sunflower production is expanding to arid regions, tolerance to abiotic stresses as drought, low temperatures and salinity arises as one of the main constrains nowadays. Differential organ-specific sunflower ESTs (expressed sequence tags) were previously generated by a subtractive hybridization method that included a considerable number of putative abiotic stress associated sequences. The objective of this work is to analyze concerted gene expression profiles of organ-specific ESTs by fluorescence microarray assay, in response to high sodium chloride concentration and chilling treatments with the aim to identify and follow up candidate genes for early responses to abiotic stress in sunflower.ResultsAbiotic-related expressed genes were the target of this characterization through a gene expression analysis using an organ-specific cDNA fluorescence microarray approach in response to high salinity and low temperatures. The experiment included three independent replicates from leaf samples. We analyzed 317 unigenes previously isolated from differential organ-specific cDNA libraries from leaf, stem and flower at R1 and R4 developmental stage. A statistical analysis based on mean comparison by ANOVA and ordination by Principal Component Analysis allowed the detection of 80 candidate genes for either salinity and/or chilling stresses. Out of them, 50 genes were up or down regulated under both stresses, supporting common regulatory mechanisms and general responses to chilling and salinity. Interestingly 15 and 12 sequences were up regulated or down regulated specifically in one stress but not in the other, respectively. These genes are potentially involved in different regulatory mechanisms including transcription/translation/protein degradation/protein folding/ROS production or ROS-scavenging. Differential gene expression patterns were confirmed by qRT-PCR for 12.5% of the microarray candidate sequences.ConclusionEighty genes isolated from organ-specific cDNA libraries were identified as candidate genes for sunflower early response to low temperatures and salinity. Microarray profiling of chilling and NaCl-treated sunflower leaves revealed dynamic changes in transcript abundance, including transcription factors, defense/stress related proteins, and effectors of homeostasis, all of which highlight the complexity of both stress responses. This study not only allowed the identification of common transcriptional changes to both stress conditions but also lead to the detection of stress-specific genes not previously reported in sunflower. This is the first organ-specific cDNA fluorescence microarray study addressing a simultaneous evaluation of concerted transcriptional changes in response to chilling and salinity stress in cultivated sunflower.

DArT markers: diversity analyses and mapping in Sorghum bicolor.

BackgroundThe sequential nature of gel-based marker systems entails low throughput and high costs per assay. Commonly used marker systems such as SSR and SNP are also dependent on sequence information. These limitations result in high cost per data point and significantly limit the capacity of breeding programs to obtain sufficient return on investment to justify the routine use of marker-assisted breeding for many traits and particularly quantitative traits. Diversity Arrays Technology (DArT™) is a cost effective hybridisation-based marker technology that offers a high multiplexing level while being independent of sequence information. This technology offers sorghum breeding programs an alternative approach to whole-genome profiling. We report on the development, application, mapping and utility of DArT™ markers for sorghum germplasm.ResultsA genotyping array was developed representing approximately 12,000 genomic clones using PstI+BanII complexity with a subset of clones obtained through the suppression subtractive hybridisation (SSH) method. The genotyping array was used to analyse a diverse set of sorghum genotypes and screening a Recombinant Inbred Lines (RIL) mapping population. Over 500 markers detected variation among 90 accessions used in a diversity analysis. Cluster analysis discriminated well between all 90 genotypes. To confirm that the sorghum DArT markers behave in a Mendelian manner, we constructed a genetic linkage map for a cross between R931945-2-2 and IS 8525 integrating DArT and other marker types. In total, 596 markers could be placed on the integrated linkage map, which spanned 1431.6 cM. The genetic linkage map had an average marker density of 1/2.39 cM, with an average DArT marker density of 1/3.9 cM.ConclusionWe have successfully developed DArT markers for Sorghum bicolor and have demonstrated that DArT provides high quality markers that can be used for diversity analyses and to construct medium-density genetic linkage maps. The high number of DArT markers generated in a single assay not only provides a precise estimate of genetic relationships among genotypes, but also their even distribution over the genome offers real advantages for a range of molecular breeding and genomics applications.

Identification of novel genes associated with conidiation in Beauveria bassiana with suppression subtractive hybridization

The conidiation of the entomopathogenic fungus Beauveria bassiana (Hyphomycete) is a complex process that involves the stage- and cell-type-specific expression of hundreds of genes. The suppression subtractive hybridization method was used to target genes involved in conidiation. Seventeen genes were cloned that potentially were involved in conidia formation. Six of them demonstrated differential expression between conidial and vegetative cultures. Sequence analysis showed three cDNA fragments had similarity to known genes involved in either cellular metabolism or cell regulatory processes. The other cDNA fragments showed low or no similarity to any genes previously described. The full-length cDNA and genomic sequence of a gene designated A43 was isolated. The A43 protein is composed of 180 amino acids and has 34% identity to a RNA-binding region-containing protein. The temporal expression pattern was consistent with the gene being involved in conidiation. The colony morphology of the A43 knock-out mutant had more floccus mycelium than the wild-type and also produced fewer conidia, indicating the A43 gene is involved in B. bassiana conidiation.

Identification of novel genes associated with conidiation in Beauveria bassiana with suppression subtractive hybridization

The conidiation of the entomopathogenic fungus Beauveria bassiana (Hyphomycete) is a complex process that involves the stage- and cell-type-specific expression of hundreds of genes. The suppression subtractive hybridization method was used to target genes involved in conidiation. Seventeen genes were cloned that potentially were involved in conidia formation. Six of them demonstrated differential expression between conidial and vegetative cultures. Sequence analysis showed three cDNA fragments had similarity to known genes involved in either cellular metabolism or cell regulatory processes. The other cDNA fragments showed low or no similarity to any genes previously described. The full-length cDNA and genomic sequence of a gene designated A43 was isolated. The A43 protein is composed of 180 amino acids and has 34% identity to a RNA-binding region-containing protein. The temporal expression pattern was consistent with the gene being involved in conidiation. The colony morphology of the A43 knock-out mutant had more floccus mycelium than the wild-type and also produced fewer conidia, indicating the A43 gene is involved in B. bassiana conidiation.

Molecular cloning and characterization of common carp ( Cyprinus carpio L.) TCRγ and CD3γ/δ chains

Partial cDNA sequences of TCRγ and CD3γ/δ were isolated from the thymus of common carp ( Cyprinus carpio L.) by the method of suppression subtractive hybridization (SSH). Subsequently the full length cDNAs of carp TCRγ and CD3γ/δ were obtained by means of 3′ RACE and 5′ RACE, respectively. The full length of carp TCRγ chain is 1368 bp and encodes 326 amino acids including a signal peptide region of 19 amino acids and a transmembrane region of 23 amino acids at the C-terminal region from aa 291 to 313. The V region of carp TCRγ contains 109 amino acids, the core motif FGXG in J segment was also found in carp TCRγ. The C region of carp TCRγ contains the characteristic CX6PX6WX45C motif. The CP region of carp TCR Cγ contains 37 amino acids. The full length of carp CD3γ/δ is 790 bp and encodes 175 amino acids including a signal peptide region of 17 amino acids and a transmembrane region of 23 amino acids from aa 93 to 115. Similar to other known CD3γ/δs, four cysteine residues in the extracellular domain and an immunoreceptor tyrosine-based activation motif ITAM (YxxL/Ix6-8YxxL/I) in the intracellular domain are also included in carp CD3γ/δ. Differing from other known CD3γ/δs, carp CD3γ/δ lacks the CXXCXE motif in the extracellular domain. RT-PCR analysis demonstrated that the expression of TCRγ gene was mainly in the thymus and gill of 6-month carp, but in 18-month carp, TCRγ gene was detected in all the examined tissues. The expression of CD3γ/δ gene was detected in all examined tissues of 6 and 18-month carp; among them, the highest expression level was in the thymus of 6-month carp. In situ hybridization showed that CD3γ/δ-expressing cells were widely distributed in the head kidney, spleen and kidney of carp, whereas in the thymus, they were densely distributed in the lymphoid outer zone and scattered in the epithelioid inner zone.

Identification and analysis of differentially expressed genes in differentiating xylem of Chinese fir (Cunninghamia lanceolata) by suppression subtractive hybridization

Wood is an important raw material for global industries with rapidly increasing demand. To isolate the genes differentially expressed during xylogenesis of Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.), we used a novel system. Forward and reverse subtracted cDNA libraries were constructed using the suppression subtractive hybridization method; for the forward library we used cDNA from the mutant Dugansha as the tester and cDNA from the wild-type clone Jurong 0 as the driver, and for the reverse library we used Jurong 0 cDNA as the tester and Dugansha cDNA as the driver. Transcriptional profiling was performed using a macroarray with 4 digoxigenin-labeled probes. We obtained 618 and 409 clones from the forward and the reverse subtracted library, respectively. A total of 405 unique expressed sequence tags (ESTs) were obtained. Forty percent of the ESTs exhibited homologies with proteins of known function and fell into 4 major classes: metabolism, cell wall biogenesis and remodeling, signal transduction, and stress. Real-time PCR was performed to confirm the results. The expression levels of 11 selected ESTs were consistent with both macroarray and real-time PCR results. The systematic analysis of genes involved in wood formation in Chinese fir provides valuable insights into the molecular mechanisms involved in xylem differentiation and is an important resource for forest research that can be directed toward understanding the genetic control of wood formation and future endeavors to modify wood and fiber properties for industrial use.

Screening of Pathogenicity-Related Genes in Bone Marrow CD4+ T Cells of Aplastic Anemia Patients.

Recent clinical and experimental data have proved that CD4+ T cells play a crucial role in the pathogenesis of aplastic anemia (AA). However, the mechanisms how CD4+ T cells in AA over-proliferate, activate, infiltrate bone marrow and damage hematopoietic cells have largely remained unknown. Therefore we adopted the suppressive subtractive hybridization (SSH) method so as to screen differentially expressed genes of bone marrow CD4+ T cells between AA patients and normal donors, which may hopefully help clear up the mystery of the pathogenesis of AA. Firstly, we selected a pair of subjects: a typical first-visit AA patient and a healthy donor of the same age and sex. Their bone marrow (BM) mononuclear cells were isolated using lymphocyte separating medium by density gradient centrifugation. After a 24-hour expansion culture, CD4+ T cells were separated with magnetic bead sorting technique. Secondly, with CD4+ T cells of the patient as “tester” and those of the normal donor as “driver”, a cDNA library was established by the SSH method. 110 clones were detected by means of PCR in the established subtractive library, which contained an inserted fragment with 200∼700bp respectively, and the positive detected rate was up to 88%. Then 10 of the resulting subtracted cDNA clones were randomly selected for a respective DNA sequencing and honologic analysis. Among 10 sequenced clones, there were 8 known genes including 2 repeated genes. Lastly, we collected BM samples of 20 AA patients and 20 healthy donors so as to validate the expression levels of differentially expressed genes from SSH library with semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR). We found, compared with normal donors, there were 6 genes among these 8 genes from SSH library up-expressed in BM CD4+ T cells of patients with AA. These genes were transducin (beta) -like 1X-linked receptor 1 (TBL1XR1) (NM024665); ATP-binding cassette, sub-family B (MDR/TAP), member 6 (NM005689); ARP2 actin-related protein 2 homolog (ACTR 2) (NM005722); zinc finger protein 561 (ZNF561) (NM152289); NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6 (NM002493); eukaryotic translation initiation factor 3, subunit 5(NM003754). These results indicate that some genes are found able to regulate functions of BM CD4+ T cells in AA, concerned with protein synthesis, biology oxidation, signal transduction and cell migration, etc. Therefore, screening and cloning these genes are helpful to elucidate the possible mechanisms of AA.

Structure, organization and expression of common carp ( Cyprinus carpio L.) SLP-76 gene

SLP-76 is an important member of the SLP-76 family of adapters, and it plays a key role in TCR signaling and T cell function. Partial cDNA sequence of SLP-76 of common carp ( Cyprinus carpio L.) was isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently, the full length cDNA of carp SLP-76 was obtained by means of 3′ RACE and 5′ RACE, respectively. The full length cDNA of carp SLP-76 was 2007 bp, consisting of a 5′-terminal untranslated region (UTR) of 285 bp, a 3′-terminal UTR of 240 bp, and an open reading frame of 1482 bp. Sequence comparison showed that the deduced amino acid sequence of carp SLP-76 had an overall similarity of 34–73% to that of other species homologues, and it was composed of an NH2-terminal domain, a central proline-rich domain, and a C-terminal SH2 domain. Amino acid sequence analysis indicated the existence of a Gads binding site R-X-X-K, a 10-aa-long sequence which binds to the SH3 domain of LCK in vitro, and three conserved tyrosine-containing sequence in the NH2-terminal domain. Then we used PCR to obtain a genomic DNA which covers the entire coding region of carp SLP-76. In the 9.2 k-long genomic sequence, twenty one exons and twenty introns were identified. RT-PCR results showed that carp SLP-76 was expressed predominantly in hematopoietic tissues, and was upregulated in thymus tissue of four-month carp compared to one-year old carp. RT-PCR and virtual northern hybridization results showed that carp SLP-76 was also upregulated in thymus tissue of GH transgenic carp at the age of four-months. These results suggest that the expression level of SLP-76 gene may be related to thymocyte development in teleosts.

Beetle immunity: Identification of immune-inducible genes from the model insect Tribolium castaneum

The red flour beetle, Tribolium castaneum, is an established genetically tractable model insect for evolutionary and developmental studies. Therefore, it may also represent a valuable model for comparative analysis of insect immunity. Here, we used the suppression subtractive hybridization method to identify Tribolium genes that are transcriptionally induced in response to injection of crude lipopolysaccharide (LPS). Determined genes encode proteins that share sequence similarities with counterparts from other insects known to mediate sensing of infection (e.g. Toll and PGRP) or to represent potential antimicrobial effectors (e.g. ferritin, c-type lysozyme, serine proteinase inhibitors, and defensins). Especially significant is the identification of thaumatin-like peptides, representing ancient antifungal peptides originally reported from plants, that are absent from the genomes of many other insects such as Drosophila, Anopheles, and Apis. We produced recombinant thaumatin-1 in bacteria and we found that it represents an antimicrobial peptide against filamentous fungi in Tribolium. Additionally, septic injury induces expression of genes involved in stress adaptation (e.g. heat-shock proteins) or insecticide resistance (e.g. cytochrome P450s) in Tribolium, suggesting that there may be crosstalk between the immune and stress responses.