Suppression Subtractive Hybridization Approach Research Articles

A novel prolactin receptor (PRLR2) in tilapia (Oreochromis mossambicus) is involved in the sea water acclimation response

A novel prolactin receptor (OmPRLR2) was identified and cloned in the euryhaline fish Oreochromis mossambicus using the suppression subtractive hybridization approach based on its response to hyperosmotic stress. OmPRLR2 protein has 55% identity with a known tilapia PRLR and 50% identity with human PRLR. Expression of OmPRLR2 was detected mainly in osmoregulatory organs (gills, intestine and kidney) but also in brain and muscle. Expression of the two tilapia PRLR differs in response to osmotic stress; OmPRLR1 expression is augmented during hypoosmotic stress but OmPRLR2 expression increased during hyperosmotic stress. Expression levels and tissue distribution of OmPRLR2 protein were quantified using specific antibodies, confirming its overexpression during hyperosmotic stress in gill cells. Stably transfected HEK‐293 cell lines overexpressing Tet‐inducible OmPRLR1 and OmPRLR2 were established. Receptors were functionally characterized using a casein promoter reporter system and through the analysis of expression of different prolactin (PRL) and growth hormone (GH) responsive genes. Specific responses of tilapia PRL177, tilapia PRL188, ovine PRL and tilapia GH were analyzed and show strong specificities for the ligands and resulting in activation of different signal transduction pathways. Additionally, cell lines expressing OmPRLR2 have increased tolerance to hyperosmotic media. Our results indicate that OmPRLR2 is a novel receptor contributing to osmosensory signal transduction in euryhaline fish.This work was supported by a grant from the National Science Foundation (IOB‐0542755).

Genes differentially expressed in Theobroma cacao associated with resistance to witches’ broom disease caused by Crinipellis perniciosa

SUMMARY The basidiomycete Crinipellis perniciosa is the causal agent of witches' broom disease of Theobroma cacao (cocoa). Hypertrophic growth of infected buds ('brooms') is the most dramatic symptom, but the main economic losses derive from pod infection. To identify cocoa genes differentially expressed during the early stages of infection, two cDNA libraries were constructed using the suppression subtractive hybridization (SSH) approach. Subtraction hybridization was conducted between cDNAs from infected shoot-tips of the susceptible genotype 'ICS 39' and the resistant 'CAB 214', in both directions. A total of 187 unique sequences were obtained, with 83 from the library enriched for the susceptible 'ICS 39' sequences, and 104 for the resistant 'CAB 214'. By homology search and ontology analyses, the identified sequences were mainly putatively categorized as belonging to 'signal transduction', 'response to biotic and abiotic stress', 'metabolism', 'RNA and DNA metabolism', 'protein metabolism' and 'cellular maintenance' classes. Quantitative reverse transcription amplification (RT-qPCR) of 23 transcripts identified as differentially expressed between genotypes revealed distinct kinetics of gene up-regulation at the asymptomatic stage of the disease. Expression induction in the susceptible 'ICS 39' in response to C. perniciosa was delayed and limited, while in 'CAB 214' there was a quicker and more intense reaction, with two peaks of gene induction at 48 and 120 h after inoculation, corresponding to morphological and biochemical changes previously described during colonization. Similar differences in gene induction were validated for another resistant genotype ('CAB 208') in an independent experiment. Validation of these genes corroborated similar hypothetical mechanisms of resistance described in other pathosystems.

HIGH-THROUGHPUT APPROACHES TO THE IDENTIFICATION OF CITRUS GENES INVOLVED IN FRUIT RESPONSE TO PENICILLIUM DIGITATUM INFECTION

ISHS International Symposium on Biotechnology of Temperate Fruit Crops and Tropical Species HIGH-THROUGHPUT APPROACHES TO THE IDENTIFICATION OF CITRUS GENES INVOLVED IN FRUIT RESPONSE TO PENICILLIUM DIGITATUM INFECTION

Identification of New Potential Regulators of the Medicago truncatula—Sinorhizobium meliloti Symbiosis Using a Large-Scale Suppression Subtractive Hybridization Approach

We set up a large-scale suppression subtractive hybridization (SSH) approach to identify Medicago truncatula genes differentially expressed at different stages of the symbiotic interaction with Sinorhizobium meliloti, with a particular interest for regulatory genes. We constructed 7 SSH libraries covering successive stages from Nod factor signal transduction to S. meliloti infection, nodule organogenesis, and functioning. Over 26,000 clones were differentially screened by two rounds of macroarray hybridizations. In all, 3,340 clones, corresponding to genes whose expression was potentially affected, were selected, sequenced, and ordered into 2,107 tentative gene clusters, including 767 MtS clusters corresponding to new M. truncatula genes. In total, 52 genes encoding potential regulatory proteins, including transcription factors (TFs) and other elements of signal transduction cascades, were identified. The expression pattern of some of them was analyzed by quantitative reverse-transcription polymerase chain reaction in wild-type and in Nod- M. truncatula mutants blocked before or after S. meliloti infection. Three genes, coding for TFs of the bHLH and WRKY families and a C2H2 zinc-finger protein, respectively, were found to be upregulated, following S. meliloti inoculation, in the infection-defective mutant lin, whereas the bHLH gene also was expressed in the root-hair-curling mutant hcl. The potential role of these genes in early symbiotic steps is discussed.

Use of suppression subtractive hybridization approach to identify genes differentially expressed during early banana fruit development undergoing changes in ethylene responsiveness

Little is known about the early green developmental stage of banana fruit before the commercial harvesting stage. In this study, we demonstrate that banana fruit (cv Cavendish) grown under our pedoclimatical conditions undergoes changes in ethylene responsiveness between 40 (immature fruit unable to respond to ethylene), 60 and 90 DAF (days after flowering; early and late mature fruit able to respond to ethylene, respectively). Further, we have combined subtractive suppression hybridization (SSH) and macro-array hybridization to construct four different SSH libraries comprising a total of 3072 clones and identify 876 clones that are differentially expressed during fruit ripening. Some of these positive clones were partially sequenced to generate ESTs. Sequence analysis revealed that 163 clones on 177 (92%) presented a high similarity with different genes in the database and were related to various plant mechanisms. Northern blot analysis and real-time quantitative PCR conducted on 24 selected clones showed that the subtraction worked properly and led to get more insights into the early green developmental stages of banana. These genes will contribute to increase pools of public EST collections of banana for identification of candidate genes and the providing of molecular markers usable to improve banana fruit quality throughout conventional breeding or biotechnology approaches.

Identification and pathway analysis of immediate hyperosmotic stress responsive molecular mechanisms in tilapia ( Oreochromis mossambicus) gill

Salinity is a major environmental factor that strongly influences cellular and organismal function. We have used the euryhaline fish Oreochromis mossambicus to identify and annotate immediate hyperosmotic stress responsive molecular mechanisms and biological processes in gill epithelial cells. Using a suppression subtractive hybridization (SSH) approach, we have identified and cloned 20 novel immediate early genes whose mRNAs are induced in gill epithelial cells 4 h after transfer of fish from freshwater (FW) to seawater (SW). Full-length or partial sequences of open reading frames (ORFs) were obtained using the rapid amplification of cDNA ends (RACE) technique. Kinetics of induction was analyzed for all hyperosmotic stress-induced genes. Most genes show a robust transient increase in mRNA abundance characteristic of immediate early stress response genes with peak levels observed between 2 and 8 h after seawater transfer. The newly identified genes were classified according to their sequence similarity with other vertebrate homologs and based on their predicted functions. Pathway analysis revealed that more than half of the identified immediate hyperosmotic stress genes interact closely within a cellular stress response signaling network. Moreover, the 20 genes cluster together in six molecular processes that are rapidly activated in tilapia gills upon salinity transfer. These processes are (1) stress response signal transduction, (2) compatible organic osmolyte accumulation, (3) energy metabolism, (4) lipid transport and cell membrane protection, (5) actin-based cytoskeleton dynamics, and (6) protein and mRNA stability. Our identification and analysis of a set of novel osmo-responsive tilapia genes provides insight into critical physiological processes and pathways constituting the hyperosmotic stress adaptation program in gill epithelial cells of euryhaline fishes.

Differential screening reveals genes differentially expressed in low- and high-virulence near-isogenic Phytophthora sojae lines

To explore the molecular mechanisms involved in virulence variations in Phytophthora sojae, the low-virulence isolate PS2 was inoculated successively on a resistant soybean ( Glycine max) cultivar. After 14 successive inoculations, a high-virulence progeny, termed PS2-vir, was obtained and demonstrated to exhibit lower oospore production. DNA fingerprinting revealed no large-scale DNA differences in PS2 and PS2-vir. A suppression subtractive hybridization (SSH) approach was developed to investigate differences in gene expression between PS2 and PS2-vir in the early stages of soybean infection. Of the 323 sequences chosen for examination, 74 putative unigenes were identified that exhibit high expression in PS2-vir. These sequences are predicted to encode proteins involved in energy production, protein biosynthesis, cell signaling, cell-wall biogenesis, and transcription regulation. Ten clones were selected for temporal expression analysis using RT-PCR based on the results of the dot-blot screens. The possible genetic mechanisms involved in these phenomena are discussed.

Identification of differential gene expression in in vitro FSH treated pig granulosa cells using suppression subtractive hybridization

FSH, which binds to specific receptors on granulosa cells in mammals, plays a key role in folliculogenesis. Its biological activity involves stimulation of intercellular communication and upregulation of steroidogenesis, but the entire spectrum of the genes regulated by FSH has yet to be fully characterized.In order to find new regulated transcripts, however rare, we have used a Suppression Subtractive Hybridization approach (SSH) on pig granulosa cells in primary culture treated or not with FSH. Two SSH libraries were generated and 76 clones were sequenced after selection by differential screening. Sixty four different sequences were identified, including 3 novel sequences. Experiments demonstrated the presence of 25 regulated transcripts.A gene ontology analysis of these 25 genes revealed (1) catalytic; (2) transport; (3) signal transducer; (4) binding; (5) anti-oxidant and (6) structural activities. These findings may deepen our understanding of FSH's effects. Particularly, they suggest that FSH is involved in the modulation of peroxidase activity and remodelling of chromatin.

Stolbur Phytoplasma Genome Survey Achieved Using a Suppression Subtractive Hybridization Approach with High Specificity

Phytoplasmas are unculturable bacterial plant pathogens transmitted by phloem-feeding hemipteran insects. DNA of phytoplasmas is difficult to purify because of their exclusive phloem location and low abundance in plants. To overcome this constraint, suppression subtractive hybridization (SSH) was modified and used to selectively amplify DNA of the stolbur phytoplasma infecting a periwinkle plant. Plasmid libraries were constructed, and the origins of the DNA inserts were verified by hybridization and PCR screenings. After a single round of SSH, there was still a significant level of contamination with plant DNA (around 50%). However, the modified SSH, which included a second round of subtraction (double SSH), resulted in an increased phytoplasma DNA purity (97%). Results validated double SSH as an efficient way to produce a genome survey for microbial agents unavailable in culture. Assembly of 266 insert sequences revealed 181 phytoplasma genetic loci which were annotated. Comparative analysis of 113 kbp indicated that among 217 protein coding sequences, 83% were homologous to "Candidatus Phytoplasma asteris" (OY-M strain) genes, with hits widely distributed along the chromosome. Most of the stolbur-specific SSH sequences were orphan genes, with the exception of two partial coding sequences encoding proteins homologous to a mycoplasma surface protein and riboflavin kinase.

A Suppression Subtractive Hybridization Approach Reveals Niche-Specific Genes That May Be Involved in Predator Avoidance in Marine Synechococcus Isolates

Picocyanobacteria of the genus Synechococcus are important contributors to marine primary production and are ubiquitous in the world's oceans. This genus is genetically diverse, and at least 10 discrete lineages or clades have been identified phylogenetically. However, little if anything is known about the genetic attributes which characterize particular lineages or are unique to specific strains. Here, we used a suppression subtractive hybridization (SSH) approach to identify strain- and clade-specific genes in two well-characterized laboratory strains, Synechococcus sp. strain WH8103 (clade III) and Synechococcus sp. strain WH7803 (clade V). Among the genes that were identified as potentially unique to each strain were genes encoding proteins that may be involved in specific predator avoidance, including a glycosyltransferase in strain WH8103 and a permease component of an ABC-type polysaccharide/polyol phosphate export system in WH7803. During this work the genome of one of these strains, WH7803, became available. This allowed assessment of the number of false-positive sequences (i.e., sequences present in the tester genome) present among the SSH-enriched sequences. We found that approximately 9% of the WH8103 sequences were potential false-positive sequences, which demonstrated that caution should be used when this technology is used to assess genomic differences in genetically similar bacterial strains.

Caste-based differences in gene expression in the polyembryonic wasp Copidosoma floridanum

The polyembryonic parasitoid Copidosoma floridanum produces two larval castes, soldiers and reproductives, during development within its host. Soldier larvae defend the brood against competitors while reproductive larvae develop into adult wasps. As with other caste-forming insects, the distinct morphological and behavioral features of soldier and reproductive larvae likely involve differential gene expression. In this study we used a bi-directional suppression subtractive hybridization (SSH) approach to isolate differentially expressed genes from C. floridanum soldier and reproductive larvae. We isolated 230 novel expressed sequence tags (ESTs) from the two subtractions (114 soldier/116 reproductive ESTs). Among these ESTs were sequences with significant similarity to genes coding for serine proteinases, proteinase inhibitors, odorant-binding and chemosensory proteins, and cuticular proteins. Also, three novel genes were isolated that resemble one another in conceptual translation and share the cysteine spacing pattern of short scorpion toxins and insect defensins. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of 20 ESTs from the two libraries indicated that 85% were differentially expressed in one caste or the other. We conclude that our SSH strategy was effective in identifying a number of genes differentially expressed in soldier and reproductive larvae and that several of these genes will be useful in characterizing caste-specific gene networks in C. floridanum.

Genome-wide analysis of differentially expressed genes from Penicillium chrysogenum grown with a repressing or a non-repressing carbon source

Penicillium chrysogenum is an economically important ascomycete used as industrial producer of penicillin. However, with the exception of penicillin biosynthesis genes, little attention has been paid to the genetics of other aspects of the metabolism of this fungus. In this article we describe the first attempt of systematic analysis of expressed genes in P. chrysogenum, using a suppression subtractive hybridization approach to clone and identify sequences of genes differentially expressed in media with glucose or lactose as carbon source (penicillin-repressing or non-repressing conditions). A total of 167 clones were analysed, 95 from the glucose condition and 72 from the lactose condition. Genes differentially expressed in the glucose condition encode mainly proteins involved in the mitochondrial electron transport chain and primary metabolism. Genes expressed differentially in lactose-containing medium include genes for secondary metabolism (pcbC, isopenicillin N synthase), different hydrolases and a gene encoding a putative hexose transporter or sensor. The results provided information on how the metabolism of this fungus adapts to different carbon sources. The expression patterns of some of the genes support the hypothesis that glucose induces higher rates of respiration in P. chrysogenum while repressing secondary metabolism.

Comparison of transcript profiles between near-isogenic maize lines in association with SCMV resistance based on unigene-microarrays

The molecular mechanisms underlying the development and progression of sugarcane mosaic virus (SCMV) infection in maize are poorly understood. A transcript profiling study based on maize unigene-microarrays was conducted to identify genes associated with SCMV resistance in the near-isogenic line (NIL) pair F7 + (SCMV resistant) and F7 (susceptible). Altogether, 497 differentially expressed genes were identified in 4 comparisons addressing constitutive genetic differences, inducible genetic differences, compatible reaction, and incompatible reaction. Compared to a suppression subtractive hybridization (SSH) approach on the same materials, expression patterns of microarray-ESTs and SSH-ESTs were consistent for the same comparisons despite technical discrepancies. Pathogen-induced transcripts were underrepresented on the unigene-microarray, consequently fewer microarray-ESTs (45.8%) were classified into pathogenesis-related categories than SSH-ESTs (60.5%). Moreover, fewer microarray-ESTs (4) co-segregated with Scmv QTL than SSH-ESTs (18). However, our results demonstrate that the microarray experiments complement the SSH-macroarray studies. Good candidate genes (CGs) associated with SCMV resistance can be chosen from three classes: (i) positional CGs co-localized with major Scmv QTL, (ii) functional CGs exhibiting the homology to pathogenesis-related genes, or (iii) differentially expressed ESTs showing consistent expression pattern in both approaches.

Mycorrhiza helper bacteriumStreptomycesAcH 505 induces differential gene expression in the ectomycorrhizal fungusAmanita muscaria

The interaction between the mycorrhiza helper bacteria Streptomyces nov. sp. 505 (AcH 505) and Streptomyces annulatus 1003 (AcH 1003) with fly agaric (Amanita muscaria) and spruce (Picea abies) was investigated. The effects of both bacteria on the mycelial growth of different ectomycorrhizal fungi, on ectomycorrhiza formation, and on fungal gene expression in dual culture with AcH 505 were determined. The fungus specificities of the streptomycetes were similar. Both bacterial species showed the strongest effect on the growth of mycelia at 9 wk of dual culture. The effect of AcH 505 on gene expression of A. muscaria was examined using the suppressive subtractive hybridization approach. The responsive fungal genes included those involved in signalling pathways, metabolism, cell structure, and the cell growth response. These results suggest that AcH 505 and AcH 1003 enhance mycorrhiza formation mainly as a result of promotion of fungal growth, leading to changes in fungal gene expression. Differential A. muscaria transcript accumulation in dual culture may result from a direct response to bacterial substances.

Identification of Pseudomonas aeruginosa-induced genes in human mast cells using suppression subtractive hybridization: up-regulation of IL-8 and CCL4 production

Mast cells have recently been found to be a major player in the host defence against bacterial infection through secretion of potent mediators. Identification of bacteria-induced mast cell mediators and intracellular signalling molecules involved during bacterial infection remains a major area of investigation. Recently we found an active interaction between mast cells and Pseudomonas aeruginosa bacteria. To further characterize specific genes in mast cells modulated by P. aeruginosa, we used a new approach for the study of mast cell-bacteria interaction; the suppression subtractive hybridization (SSH). SSH approach does not require a prerequisite knowledge of target genes and does not rely on the availability of the assay reagents for the specific genes. Using SSH, 94 clones were randomly selected from the subtracted cDNA library for differential screening leading to the identification of 14 P. aeruginosa-up-regulated transcripts. Sequence analysis revealed that expression of IL-1, IL-8 and CCL4 was increased by human mast cells after P. aeruginosa infection. Increased production of IL-1, IL-8 and CCL4 was confirmed at the protein levels. In addition, sequence analysis of the clones also suggests that ribosomal protein S3 and cytochrome b as well as additional 4 uncharacterized genes may potentially be involved in P. aeruginosa pathogenesis. Thus, SSH is an effective approach by identifying potential molecular targets for the study of mechanisms involved in P. aeruginosa and mast cell interaction.

Identification of genes regulated by oleic acid in Jurkat cells by suppressive subtractive hybridization analysis

In this study, the effect of oleic acid (50 μM) on gene expression of Jurkat cells (human T lymphocytes cell line) was examined using the suppressive subtractive hybridization approach. This technique allowed us to identify genes with higher or lower expression after cell treatment with oleic acid as compared to untreated cells. Oleic acid upregulated the expression of the translation elongation factor alpha 1 and ATP synthase 8 and downregulated gp96 (human tumor rejection antigen gp96), heat-shock protein 60 and subtilisin-like protein 4. These results suggest that oleic acid, at plasma physiological concentration, can regulate the expression of important genes to maintain the machinery that ensures cell functioning.

Identification of Botrytis cinerea genes up-regulated during infection and controlled by the Galpha subunit BCG1 using suppression subtractive hybridization (SSH).

The Galpha subunit BCG1 plays an important role during the infection of host plants by Botrytis cinerea. Delta bcg1 mutants are able to conidiate, penetrate host leaves, and produce small primary lesions. However, in contrast to the wild type, the mutants completely stop invasion of plant tissue at this stage; secondary lesions have never been observed. Suppression subtractive hybridization (SSH) was used to identify fungal genes whose expression on the host plant is specifically affected in bcg1 mutants. Among the 22 differentially expressed genes, we found those which were predicted to encode proteases, enzymes involved in secondary metabolism, and others encoding cell wall-degrading enzymes. All these genes are highly expressed during infection in the wild type but not in the mutant. However, the genes are expressed in both the wild type and the mutant under certain conditions in vitro. Most of the BCG1-controlled genes are still expressed in adenylate cyclase (bac) mutants in planta, suggesting that BCG1 is involved in at least one additional signaling cascade in addition to the cAMP-depending pathway. In a second SSH approach, 1,500 clones were screened for those that are specifically induced by the wild type during the infection of bean leaves. Of the 22 BCG1-controlled genes, 11 also were found in the in planta SSH library. Therefore, SSH technology can be successfully applied to identify target genes of signaling pathways and differentially expressed genes in planta.

Identification of differentially expressed genes in colorectal adenoma compared to normal tissue by suppression subtractive hybridization

Altered gene expression that results in unrestricted growth and loss of differentiation is a hallmark of neoplastic cells. The goal of the present study was to determine early changes in gene expression profiles during colorectal carcinogenesis with emphasis on those genes whose expression is upregulated in adenoma tissue compared to adjacent normal colon. Using the suppression subtractive hybridization (SSH) approach on enriched colonic crypts we identified 14 candidate genes showing increased expression levels in the original adenoma sample. Subsequent dot blot, Northern blot, standard and real-time RT-PCR analysis confirmed increased transcript levels of ten genes in various subsets of colorectal adenoma and cancer samples. SERPINB5 and a yet uncharacterized HERV-H sequence showed the highest expression in adenoma and tumor samples without significant expression in the corresponding normal tissue. These genes might represent promising new candidates for colorectal cancer early detection.

Changes in gene expression during meristem activation processes in Solanum tuberosum with a focus on the regulation of an auxin response factor gene*

A suppression subtractive hybridization approach (SSH) was used to generate a cDNA library enriched in clones representing genes that are up-regulated in the potato tuber apical bud on dormancy release. The sequences of cDNAs representing 385 different genes were determined. This study focuses on the characterization of one of these cDNAs. On the basis of sequence similarity, the cDNA was identified as encoding a member of the auxin response factor family (ARF6). The expression pattern of potato ARF6 was determined by in situ hybridization. In apical tuber buds in the early stages of sprouting, relatively high levels of ARF6-specific transcripts were detected, especially in the peripheral zones of the tunica and corpus of the apical meristems. Expression was also detected in procambial and early vascular tissues, both subtending the meristem and in adjacent leaf primordia. By contrast, in dormant buds no expression of ARF6 could be detected. The expression pattern was also determined during the tuberization process; steady-state expression levels decreased c. 10-fold in the apical region as tuberization proceeded. In non-growing buds, exhibiting correlative inhibition, ARF6-specific transcript levels were relatively low, but rapidly increased when apical dominance was removed by excision of the apical bud. The effects of gibberellin and auxin on axillary bud growth and ARF6 expression are described.

Gene expression profiling of differentially expressed genes in granulosa cells of bovine dominant follicles using suppression subtractive hybridization.

Development of antral follicles beyond 3 to 4 mm in cattle appears as a wave pattern that occurs two to three times during the estrous cycle. Each wave presents a cyclic recruitment of multiple follicles at the 3- to 4-mm stage, followed by the selection of a single follicle that becomes the dominant follicle (DF). The molecular determinants involved in the follicular dominance process remain poorly understood. The objective of the current study was to compare gene expression in granulosa cells (GCs) between growing dominant follicles from Day 5 of the estrous cycle and nonselected small follicles (<or=4 mm) using the suppression subtractive hybridization (SSH) approach to identify candidate genes differentially expressed in GCs of the DF. Small follicle cDNAs were subtracted from DF cDNAs (DF-SF) and used to establish a DF GC-subtracted cDNA library. A total of 42 nonredundant cDNAs were identified. Detection of previously identified genes such as CX43, CYP19, INHBA, and SERPINE2 supported the validity of our experimental model and the use of SSH as the method of analysis. For selected genes such as ApoER2, CPD, CSPG2, 14-3-3 epsilon, NR5A2/SF2, RGN/SMP30, and SERPINE2, gene expression profiles were compared by virtual Northern blot or reverse transcriptase-polymerase chain reaction, and results confirmed an increase or induction of their mRNA in GCs of dominant follicles compared with that of small follicles. We conclude that we have identified novel genes (known and unknown) that are up-regulated in bovine GCs that may affect follicular growth, dominance, or both.