PCR-based Suppression Subtractive Hybridization Research Articles

Isolation, identification and expression analysis of salt-induced genes in Suaeda maritima, a natural halophyte, using PCR-based suppression subtractive hybridization.

BackgroundDespite wealth of information generated on salt tolerance mechanism, its basics still remain elusive. Thus, there is a need of continued effort to understand the salt tolerance mechanism using suitable biotechnological techniques and test plants (species) to enable development of salt tolerant cultivars of interest. Therefore, the present study was undertaken to generate information on salt stress responsive genes in a natural halophyte, Suaeda maritima, using PCR-based suppression subtractive hybridization (PCR-SSH) technique.ResultsForward and reverse SSH cDNA libraries were constructed after exposing the young plants to 425 mM NaCl for 24 h. From the forward SSH cDNA library, 429 high quality ESTs were obtained. BLASTX search and TIGR assembler programme revealed overexpression of 167 unigenes comprising 89 singletons and 78 contigs with ESTs redundancy of 81.8%. Among the unigenes, 32.5% were found to be of special interest, indicating novel function of these genes with regard to salt tolerance. Literature search for the known unigenes revealed that only 17 of them were salt-inducible. A comparative analysis of the existing SSH cDNA libraries for NaCl stress in plants showed that only a few overexpressing unigenes were common in them. Moreover, the present study also showed increased expression of phosphoethanolamine N-methyltransferase gene, indicating the possible accumulation of a much studied osmoticum, glycinebetaine, in halophyte under salt stress. Functional categorization of the proteins as per the Munich database in general revealed that salt tolerance could be largely determined by the proteins involved in transcription, signal transduction, protein activity regulation and cell differentiation and organogenesis.ConclusionThe study provided a clear indication of possible vital role of glycinebetaine in the salt tolerance process in S. maritima. However, the salt-induced expression of a large number of genes involved in a wide range of cellular functions was indicative of highly complex nature of the process as such. Most of the salt inducible genes, nonetheless, appeared to be species-specific. In light of the observations made, it is reasonable to emphasize that a comparative analysis of ESTs from SSH cDNA libraries generated systematically for a few halophytes with varying salt exposure time may clearly identify the key salt tolerance determinant genes to a minimum number, highly desirable for any genetic manipulation adventure.

Analysis of gene expression in rice root tips treated with R-1-α-methylbenzyl-3- p-tolylurea using PCR-based suppression subtractive hybridization

The optically active chemical 1-α-methylbenzyl-3- p-tolylurea (MBTU) shows diverse properties in plant physiology. The root growth of Cyperus paddy weeds and plants belonging to the tribe Oryzeae are inhibited enantioselectively by R-MBTU. R-MBTU’s mode of action is not yet known. Using the PCR-based suppression subtractive hybridization (SSH) technique, we aimed to elucidate the mechanisms by which R-MBTU inhibits root growth of rice ( Oryza sativa L .). Forward and reverse subtracted cDNA libraries were obtained after cloning, and these clones were analyzed by quantitative real-time RT-PCR. R-MBTU treatment resulted in down-regulated mRNA levels of several clones; putatively a NADH-dependent glutamate synthase, plastid aspartate aminotransferase, endo-1,4-β-glucanase Cel1, 26S proteasome regulatory subunit S2, and serine/threonine-protein kinase. The mRNA level of a putative histone deacetylase was up-regulated. The putative functions of these clones suggest that root growth inhibition by R-MBTU may be associated with disruption of nitrogen metabolism, amino acid biosynthesis, cellulose synthesis, and cell cycle, and repression of transcription of various genes. We discuss how and which metabolic pathways are affected in the inhibition of root growth with R-MBTU treatment.

Cthrc1 Is a Positive Regulator of Osteoblastic Bone Formation

BackgroundBone mass is maintained by continuous remodeling through repeated cycles of bone resorption by osteoclasts and bone formation by osteoblasts. This remodeling process is regulated by many systemic and local factors.Methodology/Principal FindingsWe identified collagen triple helix repeat containing-1 (Cthrc1) as a downstream target of bone morphogenetic protein-2 (BMP2) in osteochondroprogenitor-like cells by PCR-based suppression subtractive hybridization followed by differential hybridization, and found that Cthrc1 was expressed in bone tissues in vivo. To investigate the role of Cthrc1 in bone, we generated Cthrc1-null mice and transgenic mice which overexpress Cthrc1 in osteoblasts (Cthrc1 transgenic mice). Microcomputed tomography (micro-CT) and bone histomorphometry analyses showed that Cthrc1-null mice displayed low bone mass as a result of decreased osteoblastic bone formation, whereas Cthrc1 transgenic mice displayed high bone mass by increase in osteoblastic bone formation. Osteoblast number was decreased in Cthrc1-null mice, and increased in Cthrc1 transgenic mice, respectively, while osteoclast number had no change in both mutant mice. In vitro, colony-forming unit (CFU) assays in bone marrow cells harvested from Cthrc1-null mice or Cthrc1 transgenic mice revealed that Cthrc1 stimulated differentiation and mineralization of osteoprogenitor cells. Expression levels of osteoblast specific genes, ALP, Col1a1, and Osteocalcin, in primary osteoblasts were decreased in Cthrc1-null mice and increased in Cthrc1 transgenic mice, respectively. Furthermore, BrdU incorporation assays showed that Cthrc1 accelerated osteoblast proliferation in vitro and in vivo. In addition, overexpression of Cthrc1 in the transgenic mice attenuated ovariectomy-induced bone loss.Conclusions/SignificanceOur results indicate that Cthrc1 increases bone mass as a positive regulator of osteoblastic bone formation and offers an anabolic approach for the treatment of osteoporosis.

Expression profiling in HcrVf2-transformed apple plants in response to Venturia inaequalis

Apple scab resistance is one of the most well-characterized plant–pathogen interactions in a woody plant species. While the HcrVf2 gene from the wild apple Malus floribunda 821 has proved capable of conferring scab resistance to the susceptible cv. Gala after genetic transformation, its identification represents only the first step in understanding the molecular mechanisms and, hence, the network of genes underlying the defence response. We used a PCR-based suppression subtractive hybridization to identify apple genes that are differentially expressed after Venturia inaequalis inoculation. Subtractive hybridization was performed between cDNA from challenged leaves of HcrVf2-resistant transgenic Gala and susceptible cv. Gala plants. A library of 523 unigenes was constructed and characterized by assigning a putative function via comparison with public databases. This set of pathogen-modulated apple genes includes many defence-related genes and is therefore an important source of information for understanding the molecular basis of the Malus–V. inaequalis interaction.

Isolation of cDNAs for hardening-induced genes from Chlorella vulgaris by suppression subtractive hybridization

Several organisms acquire freezing tolerance by altering intracellular reactions under sublethal low temperatures. Although many researchers have reported that organisms change their intracellular functions, such as transcript level and enzymatic activity, under stress conditions, acquisition mechanisms of freezing tolerance are still poorly clarified. In this study, we attempted to identify novel hardening-induced genes from Chlorella vulgaris C-27, a frost-hardy strain. A PCR-based suppression subtractive hybridization library was generated and the corresponding cDNA clones were isolated. A total of 263 unique cDNA clones were obtained and sequenced. Homology analysis showed that 64 distinct known proteins were encoded by the respective clones. The expression patterns of 29 of the genes were analyzed by using qPCR. Especially, six genes, which respectively encode two late embryogenesis abundant (LEA) proteins (HIC6 and HIC12), NADPH thioredoxin reductase (NTR), chlorophyll a/ b-binding protein (Cab), ζ-carotene desaturase, and Nip7, showed remarkable increase in transcripts over 100 times greater than those of unhardened cells. We discuss the possible contribution of the genes, which showed remarkable transcriptional increases, in acquisition of freezing tolerance of Chlorella.

Differential expression of metallothionein type-2 homologues in leaves and roots of Black pepper (Piper nigrum L)

Black pepper (Piper nigrum L.), member of the family Piperaceae is indigenous to India and is one of the most widely used spices in the world. In this paper we report the results of our attempts to identify a set of genes differentially expressed in the leaves of Piper nigrum, which could facilitate targeted engineering of this valuable crop. A PCR-based Suppression Subtractive Hybridization (SSH) technique was used to generate a leaf-specific subtracted cDNA library of Piper nigrum. A tester population of leaf cDNA was subtracted with a root derived driver cDNA. The efficiency of subtraction was confirmed by PCR analysis using the housekeeping gene actin. On sequence analysis, almost 30% of the clones showed homology to metallothionein type-2 gene. The predominance of metallothionein transcripts in the leaf was further confirmed using Real-Time PCR analyses and Northern blot. The possible role of metallothionein type-2 homologues in the leaf is discussed along with the feasibility of using SSH technique for identification of more number of tissue-specific genes from Piper nigrum.

Genome comparison ofBifidobacterium longumstrains NCC2705 and CRC-002 using suppression subtractive hybridization

Because probiotic effects are strain dependent, genomic explanations of these differences will contribute to understanding their mechanisms of action. The genomic sequence of the Bifidobacterium longum probiotic strain NCC2705 was determined, but little is known about the genetic diversity between strains of this species. Suppression subtractive hybridization (SSH) is a powerful method for generating a set of DNA fragments differing between two closely related bacterial strains. The purpose of this study was to identify genetic differences between genomes of B. longum strains NCC2705 and CRC-002 using PCR-based SSH. Strain CRC-002 produces exopolysaccharides whereas NCC2705 is not known for reliable exopolysaccharide production. Thirty-five and 30 different sequences were obtained from the SSH libraries of strains CRC-002 and NCC2705, respectively. Specific CRC-002 genes found were predicted to be involved in the biosynthesis of exopolysaccharides and metabolism of other carbohydrates, and these genes were not present in the genome of strain NCC2705. The identification of an endo-1,4-beta-xylanase gene in the CRC-002 SSH library is an important difference because xylanase genes have previously been proposed as a defining characteristic of the NCC2705 strain. The results demonstrate that the SSH technique was useful to highlight potential genes involved in complex sugar metabolism that differ between the two probiotic strains.

Identification of two novel nodule-specific genes from Astragalus sinicus L. by suppressive subtractive hybridization

To identify the genes involved in nodule formation and to increase usable molecular probes, a cDNA library of Astragalus sinicus genes specifically expressed in infected roots by Mesorhizobium huakuii 7653R is generated using a PCR-based suppressive subtractive hybridization (SSH) technique with two mRNA populations of infected and uninfected control roots. Two nodule-specific genes, AsIIC259 and AsG2511, are identified from infected roots of A. sinicus. The amino acid sequences deduced from the open reading frames (ORFs) reveal that AsIIC259 and AsG2511 encodes a polypeptide with 134 and 58 amino acids, respectively. A signal peptide sequence is predicted with high probability at the N-termini of the AsIIC259 and AsG2511. The motif searches show that the deduced polypeptide of AsIIC259 contains two N-glycosylation sites, a cAMP-and cGMP-dependent protein kinase phosphorylation site and a casein kinase II phosphorylation site. BLASTP searches reveal that AsIIC259 putative protein displays a low degree of similarity to a unique nodulin from Lupinus luteus nodules. No significant identity is displayed over the predicted polypeptides of AsG2511 with any published sequences. Virtual Northern blot and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses indicate that the two genes are expressed exclusively in inoculated roots and that their expression is 2–4 d later than that of the leghaemoglobin (Lb) gene during nodule development.

41. Research for gene expression in rice root tips treated with R-1-α-methylbenzyl-3-p-tolylurea using PCR-based suppression subtractive hybridization (SSH)

41. Research for gene expression in rice root tips treated with R-1-α-methylbenzyl-3-p-tolylurea using PCR-based suppression subtractive hybridization (SSH)

Subtractive hybridization and optical mapping of the enterotoxigenic Escherichia coli H10407 chromosome: isolation of unique sequences and demonstration of significant similarity to the chromosome of E. coli K-12

Enterotoxigenic Escherichia coli (ETEC) is a primary cause of diarrhoea in infants in developing countries and in travellers to endemic regions. While several virulence genes have been identified on ETEC plasmids, little is known about the ETEC chromosome, although it is expected to share significant homology in backbone sequences with E. coli K-12. In the absence of genomic sequence information, the subtractive hybridization method and the more recently described optical mapping technique were carried out to determine the degree of genomic variation between virulent ETEC strain H10407 and the non-pathogenic E. coli K-12 strain MG1655. In one round of PCR-based suppression subtractive hybridization, 153 fragments representing sequences unique to strain H10407 were identified. blast searches indicated that few unique sequences showed homology to known pathogenicity island genes identified in related E. coli pathogens. A total of 65 fragments contained sequences that were either linked to hypothetical proteins or showed no homology to any known sequence in the database. The remaining sequences were either phage or prophage related or displayed homology to classifiable genes that function in various aspects of bacterial metabolism. The 153 unique sequences showed variable distribution across different ETEC strains including ETEC strain B7A, which is attenuated in virulence and lacked several H10407-specific sequences. Restriction-enzyme-based optical maps of strain H10407 were compared to in silico restriction maps of strain MG1655 and related E. coli pathogens. The 5.1 Mb ETEC chromosome was approximately 500 kb greater in length than the chromosome of E. coli K-12, collinear with it and indicated several discrete regions where insertions and/or deletions had occurred relative to the chromosome of strain MG1655. No major inversions, transpositions or gross rearrangements were observed on the ETEC chromosome. Based on comparisons with known genomic sequences and related optical-map-based restriction site similarity, the sequence of the H10407 chromosome is expected to demonstrate approximately 96 % identity with that of E. coli K-12.

Phenotypical and molecular characterization of the Tomato mottle Taino virus–Nicotiana megalosiphon interaction

Tomato mottle Taino virus (ToMoTV) infection causes significant yield losses in plants of various Solanaceous species. In this study, the interaction between Nicotiana megalosiphon and ToMoTV was characterized on a phenotypical and molecular basis. In order to isolate genes that are differentially expressed during the interaction of N. megalosiphon with ToMoTV, a PCR-based suppression subtractive hybridization (SSH) was utilized. RNA dot-blot analysis confirmed induction of representative genes upon ToMoTV inoculation at different time points. Interestingly, most of the genes identified are reported here for the first time to be involved in the response of N. megalosiphon to begomovirus infection.

Suppression subtractive hybridization-mediated transcriptome analysis from multiple tissues of aspen (Populus tremuloides) altered in phenylpropanoid metabolism.

A PCR-based suppression subtractive hybridization (SSH) technique was used to identify differentially expressed genes in developing tissues of control and transgenic aspen (Populus tremuloides Michx.) with down-regulated 4CL1 (4-coumarate:coenzyme A ligase) expression and enhanced growth. A total of 11,308 expressed sequence tags (ESTs) representing 5,028 non-redundant transcripts encoding 4,224 unique proteins was obtained from shoot apex, young stem, young leaf and root tip SSH libraries. Putative functions can be assigned to 60% of these transcripts. Approximately 14% of the ESTs are not represented among the 111,000 entries already present in Populus EST databases. In general, ESTs of the metabolism class occurred at a higher frequency in control- than transgenic-enriched libraries of all tissues, whereas protein synthesis and protein fate ESTs were over-represented in meristematic tissues of transgenics where 4CL1 was relatively strongly suppressed. Among all tissues, leaves yielded the highest percentage of ESTs with either unknown protein function or insignificant similarity to other protein/DNA/EST sequences in existing databases. Of particular interest was a large number of ESTs (16%) associated with signal transduction in transgenic leaves. Among these were several leucine-rich-repeat receptor-like protein kinases with markedly elevated expression in transgenic leaves. We also identified homologs of transposable elements that were up-regulated in transgenic tissues, providing the first experimental data for active expression of DNA mobile elements in long-lived tree species.

A novel gene in the takeout gene family is regulated by hormones and nutrients in Manduca larval epidermis

A novel gene, moling, was cloned from epidermal RNA of the tobacco hornworm, Manduca sexta, using PCR-based suppression subtractive hybridization. moling belongs to a gene family that includes several lepidopteran hemolymph juvenile hormone (JH) binding proteins and takeout of Drosophila melanogaster. The mRNA first appears in the epidermis on day 0 of the fifth instar and rises to its peak expression by mid-day 2, then declines rapidly and is gone by the onset of wandering. moling is expressed exclusively in the last instar larval epidermis and not in the imaginal discs or any other tissues. Allatectomy early in the fourth instar induces precocious metamorphosis and causes the appearance of moling mRNA by 33 h. Allatectomy after the critical period for JH in the final larval molt had no effect on the timing of the onset of moling expression in the final instar but caused a more rapid up-regulation once begun. The JH mimic pyriproxifen given at the outset of the final instar suppressed the expression of moling mRNA to low levels, in both intact and allatectomized larvae. Starvation immediately after ecdysis to the fifth instar prevented the onset of expression. Thus, initiation of transcription requires both nutrient intake and decline in JH. Infusion of 20-hydroxyecdysone (20E) into ligated abdomens of day 2 fifth instar larvae and culture of the day 2 fifth instar larval abdominal epidermis with 20E in vitro both caused a rapid decline of moling mRNA. The slower and variable decline that occurred in mid-day 2 fifth instar larval epidermis in the ligated abdomens or when incubated in hormone-free medium indicated that the increase of 20E on day 2 had already initiated the decline of expression. The role of Moling may be to stabilize JH in the epidermal cell during the final intermolt when the JH esterase activity increases.

Reactive oxygen species alter gene expression in podocytes: induction of granulocyte macrophage-colony-stimulating factor.

It has been suggested that reactive oxygen radicals (ROS) play a crucial role in the pathogenesis of proteinuria and podocyte injury. It was investigated whether changes in gene expression might account for ROS-induced podocyte dysfunction. Differentiated podocytes were incubated with control media or with exogenous ROS from the xanthine/xanthine-oxidase reaction for 4 h. A PCR-based suppressive subtractive hybridization assay was applied to isolate and clone mRNAs that were differentially expressed by exogenous ROS. One differentially expressed clone was identified as the proinflammatory cytokine granulocyte macrophage-colony-stimulating factor (GM-CSF). Regulation of GM-CSF in podocytes was further studied by Northern analysis and enzyme-linked immunosorbent assay. Exogenous ROS caused a concentration-dependent, >10-fold induction of GM-CSF mRNA after 4 h. A >50-fold increase in GM-CSF protein release in podocytes that had been stimulated with ROS could be detected. Induction of GM-CSF protein was inhibited by actinomycin D, which indicated that increased mRNA transcription was involved. The ROS scavengers dimethyl-thio-urea and pyrrolidone-dithio-carbamate strongly inhibited increased GM-CSF production induced by ROS. GM-CSF release was also induced when internal ROS production was triggered with NADH, whereas H2O2 had only a small effect. GM-CSF release by podocytes was also stimulated by lipopolysaccharide (LPS), interleukin-1 (IL-1), and phorbolester (PMA). Dimethyl-thio-urea significantly inhibited the LPS-, IL-1-, and PMA-induced GM-CSF production. Activation of the transcription factor nuclear factor-kappaB (NF-kappaB) but not activator protein-1 was involved in the upregulation of ROS-induced GM-CSF production. The data indicate that GM-CSF is differentially expressed by ROS in podocytes. ROS also partially mediate the effects of PMA and IL-1 on podocyte GM-CSF production. Because GM-CSF can enhance glomerular inflammation and induces mesangial proliferation, these data might provide further insight into the mechanisms of ROS-induced glomerular injury.

Identification of genes overexpressed in small cell lung carcinoma by PCR-based suppression subtractive hybridization and cDNA microarray

Identification of genes overexpressed in small cell lung carcinoma by PCR-based suppression subtractive hybridization and cDNA microarray

Heterogeneity of Endothelial Cells: The Specialized Phenotype of Human High Endothelial Venules Characterized by Suppression Subtractive Hybridization

High endothelial venules (HEVs) are specialized postcapillary venules, found in lymphoid organs and chronically inflamed tissues, that support high levels of lymphocyte extravasation from the blood. Molecular characterization of HEV endothelial cells (HEVECs) has been hampered by difficulties in their purification and in vitro maintenance. To overcome these limitations, we developed a strategy combining the use of freshly purified HEVECs ( approximately 98% positive for the HEV-specific marker MECA-79) and the recently described polymerase chain reaction (PCR)-based cDNA subtraction cloning procedure called suppression subtractive hybridization (SSH). Subtracted probes prepared by SSH from small amounts of total RNA were used to screen a HEVEC cDNA library. This resulted in cloning of 22 cDNAs preferentially expressed in HEVECs, which encode the promiscuous chemokine receptor DARC, mitochondrial components, and matricellular proteins. The latter included hevin, thrombospondin-1, and mac25/IGFBP-rP1, which is a secreted growth factor-binding protein previously found to accumulate specifically in tumor blood vessels. Biochemical and histochemical analysis confirmed the identification of mac25 and DARC as novel markers of the HEVECs. Ultrastructural immunolocalization revealed a noticeable association of mac25 and MECA-79 antigens with microvillous processes near the endothelial cell junctions, suggesting a role for mac25 in the control of lymphocyte emigration. This study shows that PCR-based SSH is useful for cloning of differentially expressed genes in very small samples.