Subtractive Hybridization Method Research Articles

Identification of Low Temperature Inducible Genes of Lactuca sativa by Using Suppression Subtractive Hybridization Method

Identification of Low Temperature Inducible Genes of Lactuca sativa by Using Suppression Subtractive Hybridization Method

Identification and characterization of differentially expressed genes in the rice root following exogenous application of spermidine during salt stress

Salinity is a major limiting factor in crop production. Exogenous spermidine (spd) effectively ameliorates salt injury, though the underlying molecular mechanism is poorly understood. We have used a suppression subtractive hybridization method to construct a cDNA library that has identified up-regulated genes from rice root under the treatment of spd and salt. Total 175 high-quality ESTs of about 100–500 bp in length with an average size of 200 bp are isolated, clustered and assembled into a collection of 62 unigenes. Gene ontology analysis using the KEGG pathway annotation database has classified the unigenes into 5 main functional categories and 13 subcategories. The transcripts abundance has been validated using Real-Time PCR. We have observed seven different types of post-translational modifications in the DEPs. 44 transmembrane helixes are predicted in 6 DEPs. This above information can be used as first-hand data for dissecting the administrative role of spd during salinity.

An MBT domain containing anti-lipopolysaccharide factor (PtALF8) from Portunus trituberculatus is involved in immune response to bacterial challenge

Anti-lipopolysaccharide factors are effective antimicrobial peptides that can bind and neutralize lipopolysaccharide (LPS). In the present study, a new sequence encoding for ALF (designated as PtALF8) was cloned by suppression subtractive hybridization method using ovary of swimming crab Portunus trituberculatus as material. The full-length cDNA of PtALF8 consisted of 531 bp with an ORF of 348 bp encoding a peptide of 115 amino acids containing a putative signal peptide of 19 amino acids. The mature PtALF8 had a predicted molecular weight (MW) of 11.28 kDa and theoretical isoelectricpoint (pI) of 5.11. The PtALF8 contains an MBT domain which was not found in the other 7 isoforms of ALF reported in P. trituberculatus. Unlike most ALFs expressed in hemocytes, PtALF8 transcript was predominantly detected in hepatopancreas. After challenge with Vibrio alginolyticus, the temporal expression level of PtALF8 transcript in hemocytes reached the highest level at 3 h, then decreased to the lowest level at 24 h, and started to increase at 48 h. The recombinant protein showed antimicrobial and bactericidal activity against several bacteria, such as Gram-positive bacteria, Staphylococcus aureus, Micrococcus luteus and Gram-negative bacteria, V. alginolyticus, indicated that the PtALF8 isoform might play protective function against invading bacteria in P. trituberculatus.

Analysis of differential transcript expression in chickpea during compatible and incompatible interactions with Fusarium oxysporum f. sp. ciceris Race 4.

The present study reports the transcriptome analysis of resistance (WR315) and susceptible (JG62) genotypes of chickpea in response to Fusarium oxysporum f. sp. ciceris (Foc)race 4 using the method of suppression subtractive hybridization. Altogether, 162 chickpea-expressed sequence tags (ESTs) were identified from two libraries and analyzed to catalog eight functional categories. These ESTs could be assembled into 18 contigs and 144 singletons with 10 contigs and 68 singletons from compatible and 8 contigs and 70 singletons from incompatible interaction. The largest category consisted of ESTs which encode for proteins related to hypothetical proteins (22.8%), followed by energy and metabolism (20.3%)-related genes, defense and cell rescue-related genes (17.9%) and signal transduction-related genes (16%). Among them, 17.1 and 18.7% were defense-related genes in compatible and incompatible interaction, respectively. These ESTs mainly includes various putative genes related to oxidative burst, pathogenesis and secondary metabolism. Induction of putative superoxide dismutase, metallothionein, 4-coumarate-CoA ligase, heat shock proteins and cysteine proteases indicated oxidative burst after infection. The ESTs belonged to various functional categories which were directly and indirectly associated with defense signaling pathways. Quantitative and semi-quantitative polymerase chain reaction exhibited differential expression of candidate genes and detected higher levels in incompatible interaction compared to compatible interaction. The present study revealed partial molecular mechanism associated with the resistance in chickpea against Foc, which is the key to design a strategy for incorporation of resistance via either biotechnological means or introgression of resistance genes.

Analysis of Gene Expressions Associated with Response of Phenolic Metabolism of Allelopathic Rice under Barnyardgrass Stress

Allelopathic rice varieties Giza 179 and Sakha 103 were induced by exogenous salicylic acid (SA) to suppress barnyardgrass weed (BYG) and were investigated using suppression subtractive hybridization (SSH) and real-time fluorescence quantitative PCR (qRT-PCR) methods. The results explained that exogenous salicylic acid could induce the allelopathic effect of rice on BYG. The variety Giza 179 showed higher inhibitory effect than Sakha 103 variety on BYG after treated with different concentrations of salicylic acid. Fourteen genes induced by salicylic acid were obtained by SSH analysis from Giza 179 variety. These genes encode ubiquitin carrier proteins, proteins related to phenyl-propanoid metabolism, receptor-kinase proteins, antioxidant related proteins and some growth-mediating proteins. The variations among gene expressions were validated by qRT-PCR in the two rice varieties. Previous studies indicated that cytoplasmic male sterile lines IR69625A, and G46A had general combining ability for panicle weight and grain yield per plant, while the varieties Giza 179 and Giza 178 had general combining ability for grain yield per plant and seed set percentage. In addition, with regard to the variety Giza 179 which have allelopathic potential and restoring fertility genes, so we recommend by using the variety Giza 179 as parental line with the used cytoplasmic male sterile line to produce hybrid rice have highly yield and strong allelopathy

Plant endophytic Pseudomonas putida BP25 induces expression of defense genes in black pepper roots: Deciphering through suppression subtractive hybridization analysis

Black pepper associated endophytic Pseudomonas putida BP25 displayed broad spectrum antagonistic activity against a wide range of plant pathogens. The molecular responses of black pepper upon endophytic colonization by P. putida BP25 was studied using a PCR based suppression subtractive hybridization (SSH) method. The transcripts induced by endophytic P. putida BP25 in black pepper included pathogenesis related proteins such as PR-4 and PR-1; reactive oxygen species (ROS) scavenging catalase and metallothionein; stress induced glutathione S-transferase; 5-enolpyruvylshikimate-3-phosphate synthase involved in phenylpropanoid metabolism. Differential expression of transcripts was validated by quantitative real time PCR (qPCR) for selected ESTs. This information helps in understanding the ability of an endophytic strain P. putida BP25 in inducing a wide array of defence responses in black pepper roots.

Suppression subtractive hybridization identified differentially expressed genes in colorectal cancer: microRNA-451a as a novel colorectal cancer-related gene.

To investigate differentially expressed genes regulated by microRNA-451a in colorectal cancer. We detected expression of microRNA-451a in colorectal cancer samples and normal pericarcinous tissues from 68 colorectal cancer patients and the correlation between microRNA-451a and clinical features of these patients. Then, the expression of microRNA-451a in HCT116, SW620, HT29, SW480, and DLD cells was also measured. The suppression subtractive hybridization method was used with two HCT116 cell lines with overexpressing or underexpressing microRNA-451a, respectively. The most highly increased genes were screened. Their functions were predicted by gene ontology analysis. The expression ratio of microRNA-451a in colorectal cancer to pericarcinous tissues was 0.37. Expression of microRNA-451a was decreased in HCT116, SW620, HT29, SW480, and DLD cells. In our suppression subtractive hybridization library, expression of seven genes was the most highly increased when underexpressing microRNA-451a. They were BCAP31, EEF1A1, CDC20, WDR6, TUFM, RPL13, and RPL7A. Expression of DKK1, PSME1, NDUFA3, and GNB2 was most highly increased when overexpressing microRNA-451a. Gene ontology analysis showed that the main functions of these genes were associated with translational elongation, protein localization to the endoplasmic reticulum, translation, poly(A) RNA binding, negative regulations of Wnt signaling pathway, and so on. MicroRNA-451a was demonstrated to be downregulated in colorectal cancer patient tissues whose target genes were analyzed and functions were predicted by suppression subtractive hybridization.

Full-length normalization subtractive hybridization analysis provides new insights into sexual precocity and ovarian development of red swamp crayfish Procambarus clarkii

The red swamp crayfish Procambarus clarkii is an important freshwater economic crayfish in Chinese inland due to its high market value and consumer demand. The sexual precocity in this species has received considerable attention in recent years because more and more individuals matured at small sizes, which devalues the commercial production. In this study, by the full-length normalization subtractive hybridization (FNSH) method we identified 16 differently expressed sequences between precocious and normal grown P. clarkii ovaries. Of the down-regulated sequences in precocious ovaries, one encodes a peptide which shared the highest identity to C1q-like family and contained a typical C1q domain at the C-terminal, thus it was named PcC1q-like. QRT-PCR analysis showed that PcC1q-like was transcribed specifically in ovary, with higher expression level in vitellogenic stages. In situ hybridization exhibited specific expression of PcC1q-like in follicular cells around the vitellogenic oocytes. Knockdown of PcC1q-like by injection of double strand RNA (dsRNA) resulted in disruption of follicular cells, decreased transcripts of vitellogenin (Vg) and vitellogenin receptor (VgR), and reduced progesterone content. These results suggest that PcC1q-like may promote ovarian development by stimulating the synthesis and transportation of vitellogenin into oocytes. These data provide insights into the molecular regulatory mechanisms of sexual precocity and ovarian development in crustaceans. Statement of relevanceThe red swamp crayfish Procambarus clarkii is one of the most important freshwater aquaculture species in Chinese inland. We screened 16 sequences that are potentially involved in sexual precocity of P. clarkii. One candidate was demonstrated to be a novel ovary-specific C1q-like gene of P. clarkii, thus named PcC1q-like. Knockdown of PcC1q-like resulted in disruption of follicular cells, decreased transcripts of Vg and VgR, and reduced progesterone, suggesting its essential role in vitellogenic ovarian development of P. clarkii.

Molecular changes in skin pigmented lesions of the coral trout Plectropomus leopardus

A high prevalence of skin pigmented lesions of 15% was recently reported in coral trout Plectropomus leopardus, a commercially important marine fish, inhabiting the Great Barrier Reef. Herein, fish were sampled at two offshore sites, characterised by high and low lesion prevalence. A transcriptomic approach using the suppressive subtractive hybridisation (SSH) method was used to analyse the differentially expressed genes between lesion and normal skin samples. Transcriptional changes of 14 genes were observed in lesion samples relative to normal skin samples. These targeted genes encoded for specific proteins which are involved in general cell function but also in different stages disrupted during the tumourigenesis process of other organisms, such as cell cycling, cell proliferation, skeletal organisation and cell migration. The results highlight transcripts that are associated with the lesion occurrence, contributing to a better understanding of the molecular aetiology of this coral trout skin disease.

Applying Subtractive Hybridization Technique to Enrich and Amplify Tumor-Specific Transcripts of Esophageal Squamous Cell Carcinoma

Subtractive hybridization (SH) as an efficient and powerful approach can be applied to isolate differentially expressed transcripts as well as detect of involved mRNAs in various cellular processes, particularly diseases and malignancies. This procedure leads to the enrichment of specific low copy transcripts of tumor cells. Having developed a new approach for SH to isolate tumor specific transcripts, we facilitated discovery of uniquely expressed genes in esophageal squamous cell carcinoma (ESCC). Total RNA was extracted from the fresh tumoral and their adjacent normal tissues, and purified using the Switch Mechanism At the 5' end of Reverse Transcript (SMART) method. Following cDNA synthesis of normal mRNAs using magnetic beads, it was hybridized with tumor mRNAs. To enhance efficiency of subtraction, hybridization was repeated three rounds. Finally, amplification of subtracted tumor-specific transcripts was carried out using in vitro transcription. The subtracted tumoral mRNAs was analyzed quantitatively using real-time PCR for both tumor-specific and housekeeping genes. The subtracted mRNA was confirmed as tumor-specific mRNA pool using RT-PCR and quantitative real-time PCR assessment. The elevated level of tumor-specific transcripts such as MAGE-A4 and CD44 as well as declined copy number of housekeeping genes such as GAPDH, β actin and β2-microglobulin, were confirmed in subtracted tumoral mRNA. The presence of tumor genes was confirmed after the SH procedure. The designed SH method in combination with SMART technique can isolate and amplify high quality tumor-specific transcripts even from small amount of tumor tissues. Removal of common transcripts from the extracted tumoral mRNAs using SH, leads to the enrichment of tumor-specific transcripts. The isolated transcripts are of interest because of their probable roles in ESCC progression and development. In addition, these tumor-specific mRNAs can be applied for future vaccine cancer studies.

A subtracted cDNA library identifies genes up-regulated during PHOT1-mediated early step of de-etiolation in tomato (Solanum lycopersicum L.)

BackgroundDe-etiolation is the switch from skoto- to photomorphogenesis, enabling the heterotrophic etiolated seedling to develop into an autotrophic plant. Upon exposure to blue light (BL), reduction of hypocotyl growth rate occurs in two phases: a rapid inhibition mediated by phototropin 1 (PHOT1) within the first 30–40 min of illumination, followed by the cryptochrome 1 (CRY1)-controlled establishment of the steady-state growth rate. Although some information is available for CRY1-mediated de-etiolation, less attention has been given to the PHOT1 phase of de-etiolation.ResultsWe generated a subtracted cDNA library using the suppression subtractive hybridization method to investigate the molecular mechanisms of BL-induced de-etiolation in tomato (Solanum lycopersicum L.), an economically important crop. We focused our interest on the first 30 min following the exposure to BL when PHOT1 is required to induce the process. Our library generated 152 expressed sequence tags that were found to be rapidly accumulated upon exposure to BL and consequently potentially regulated by PHOT1. Annotation revealed that biological functions such as modification of chromatin structure, cell wall modification, and transcription/translation comprise an important part of events contributing to the establishment of photomorphogenesis in young tomato seedlings. Our conclusions based on bioinformatics data were supported by qRT-PCR analyses the specific investigation of V-H+-ATPase during de-etiolation in tomato.ConclusionsOur study provides the first report dealing with understanding the PHOT1-mediated phase of de-etiolation. Using subtractive cDNA library, we were able to identify important regulatory mechanisms. The profound induction of transcription/translation, as well as modification of chromatin structure, is relevant in regard to the fact that the entry into photomorphogenesis is based on a deep reprograming of the cell. Also, we postulated that BL restrains the cell expansion by the rapid modification of the cell wall.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2613-6) contains supplementary material, which is available to authorized users.

Activation of Shikimate, Phenylpropanoid, Oxylipins, and Auxin Pathways in Pectobacterium carotovorum Elicitors-Treated Moss.

Plants have developed complex defense mechanisms to cope with microbial pathogens. Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) are perceived by pattern recognition receptors (PRRs), leading to the activation of defense. While substantial progress has been made in understanding the activation of plant defense by PAMPs and DAMPs recognition in tracheophytes, far less information exists on related processes in early divergent plants like mosses. The aim of this study was to identify genes that were induced in P. patens in response to elicitors of Pectobacterium carotovorum subsp. carotovorum, using a cDNA suppression subtractive hybridization (SSH) method. A total of 239 unigenes were identified, including genes involved in defense responses related to the shikimate, phenylpropanoid, and oxylipin pathways. The expression levels of selected genes related to these pathways were analyzed using quantitative RT-PCR, confirming their rapid induction by P.c. carotovorum derived elicitors. In addition, P. patens induced cell wall reinforcement after elicitor treatment by incorporation of phenolic compounds, callose deposition, and elevated expression of Dirigent-like encoding genes. Small molecule defense markers and phytohormones such as cinnamic acid, 12-oxo-phytodienoic acid, and auxin levels all increased in elicitor-treated moss tissues. In contrast, salicylic acid levels decreased while abscisic acid levels remained unchanged. P. patens reporter lines harboring an auxin-inducible promoter fused to β-glucuronidase revealed GUS activity in protonemal and gametophores tissues treated with elicitors of P.c. carotovorum, consistent with a localized activation of auxin signaling. These results indicate that P. patens activates the shikimate, phenylpropanoid, oxylipins, and auxin pathways upon treatment with P.c. carotovorum derived elicitors.

Research progress of multi-functional cancer killing cytokine, interleukin-24 in tumors

Interleukin-24 (IL-24) is a member of IL-10 family, which was cloned from human melanoma cells by the method of subtractive hybridization. IL-24 can inhibit the growth of tumor cells and angiogenesis and enhance radiation sensitivity and immune adjustment with non-toxic effects on normal cells. As a kind of cytokine with multiple anti-tumor functions, IL-24 will become a new weapon of gene therapy against cancer. The application and mechanism of IL-24 in the treatment of cancer will be discussed in this paper. Key words: Interleukin-24; Neoplasms; Apoptosis; Gene

Suppression Subtractive Hybridization analysis provides new insights into the tomato (Solanum lycopersicum L.) response to the plant probiotic microorganism Trichoderma longibrachiatum MK1

Trichoderma species include widespread rhizosphere-colonising fungi that may establish an opportunistic interaction with the plant, resulting in growth promotion and/or increased tolerance to biotic and abiotic stresses. For this reason, Trichoderma-based formulations are largely used in agriculture to improve yield while reducing the application of agro-chemicals. By using the Suppression Subtractive Hybridization method, we identified molecular mechanisms activated during the in vitro interaction between tomato (Solanum lycopersicum L.) and the selected strain MK1 of Trichoderma longibrachiatum, and which may participate in the stimulation of plant growth and systemic resistance. Screening and sequence analysis of the subtractive library resulted in forty unique transcripts. Their annotation in functional categories revealed enrichment in cell defence/stress and primary metabolism categories, while secondary metabolism and transport were less represented. Increased transcription of genes involved in defence, cell wall reinforcement and signalling of reactive oxygen species suggests that improved plant pathogen resistance induced by T. longibrachiatum MK1 in tomato may occur through stimulation of the above mechanisms. The array of activated defence-related genes indicates that different signalling pathways, beside the jasmonate/ethylene-dependent one, collaborate to fine-tune the plant response. Our results also suggest that the growth stimulation effect of MK1 on tomato may involve a set of genes controlling protein synthesis and turnover as well as energy metabolism and photosynthesis. Transcriptional profiling of several defence-related genes at different time points of the tomato–Trichoderma interaction, and after subsequent inoculation with the pathogen Botrytis cinerea, provided novel information on genes that may specifically modulate the tomato response to T. longibrachiatum, B. cinerea or both.

Cadmium induces cadmium-tolerant gene expression in the filamentous fungus Trichoderma harzianum

The filamentous fungus Trichoderma harzianum, strain IMI 393899, was able to grow in the presence of the heavy metals cadmium and mercury. The main objective of this research was to study the molecular mechanisms underlying the tolerance of the fungus T. harzianum to cadmium. The suppression subtractive hybridization (SSH) method was used for the characterization of the genes of T. harzianum implicated in cadmium tolerance compared with those expressed in the response to the stress induced by mercury. Finally, the effects of cadmium exposure were also validated by measuring the expression levels of the putative genes coding for a glucose transporter, a plasma membrane ATPase, a Cd(2+)/Zn(2+) transporter protein and a two-component system sensor histidine kinase YcbA, by real-time-PCR. By using the aforementioned SSH strategy, it was possible to identify 108 differentially expressed genes of the strain IMI 393899 of T. harzianum grown in a mineral substrate with the addition of cadmium. The expressed sequence tags identified by SSH technique were encoding different genes that may be involved in different biological processes, including those associated to primary and secondary metabolism, intracellular transport, transcription factors, cell defence, signal transduction, DNA metabolism, cell growth and protein synthesis. Finally, the results show that in the mechanism of tolerance to cadmium a possible signal transduction pathway could activate a Cd(2+)/Zn(2+) transporter protein and/or a plasma membrane ATPase that could be involved in the compartmentalization of cadmium inside the cell.

Identification of genes associated with shell color in the black-lipped pearl oyster, Pinctada margaritifera.

BackgroundColor polymorphism in the nacre of pteriomorphian bivalves is of great interest for the pearl culture industry. The nacreous layer of the Polynesian black-lipped pearl oyster Pinctada margaritifera exhibits a large array of color variation among individuals including reflections of blue, green, yellow and pink in all possible gradients. Although the heritability of nacre color variation patterns has been demonstrated by experimental crossing, little is known about the genes involved in these patterns. In this study, we identify a set of genes differentially expressed among extreme color phenotypes of P. margaritifera using a suppressive and subtractive hybridization (SSH) method comparing black phenotypes with full and half albino individuals.ResultsOut of the 358 and 346 expressed sequence tags (ESTs) obtained by conducting two SSH libraries respectively, the expression patterns of 37 genes were tested with a real-time quantitative PCR (RT-qPCR) approach by pooling five individuals of each phenotype. The expression of 11 genes was subsequently estimated for each individual in order to detect inter-individual variation. Our results suggest that the color of the nacre is partially under the influence of genes involved in the biomineralization of the calcitic layer. A few genes involved in the formation of the aragonite tablets of the nacre layer and in the biosynthesis chain of melanin also showed differential expression patterns. Finally, high variability in gene expression levels were observed within the black phenotypes.ConclusionsOur results revealed that three main genetic processes were involved in color polymorphisms: the biomineralization of the nacreous and calcitic layers and the synthesis of pigments such as melanin, suggesting that color polymorphism takes place at different levels in the shell structure. The high variability of gene expression found within black phenotypes suggests that the present work should serve as a basis for future studies exploring more thoroughly the expression patterns of candidate genes within black phenotypes with different dominant iridescent colors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1776-x) contains supplementary material, which is available to authorized users.

Identification of genes involved in spontaneous leaf color variation in Pseudosasa japonica.

Spontaneous leaf color variation in bamboo provides the opportunity to study the mechanisms of leaf color formation and the breeding of ornamental bamboos. Despite the fact that many genes are known to be involved in leaf color variation in model plants, molecular mechanisms governing natural leaf color variation in bamboo have remained obscure. This study aimed to identify the genes responsible for the occurrence of such phenomena in bamboo using the suppression subtractive hybridization (SSH) method between green and albino leaves in Pseudosasa japonica f. A total of 1062 and 1004 differentially expressed transcripts were obtained from the forward and reverse SSH libraries, respectively. Subsequently, 59 differentially expressed unigenes with potential roles in leaf color formation, predicted via computational analysis of their functional relevance, were selected for further analysis using qPCR. Ten genes, involved in photosynthesis, plastid development, and cation signal transduction, showed 2-fold changes in expression levels between green and albino leaves. Further expression pattern analyses of these genes at three developmental stages revealed much lower expression abundance of Lhca1-encoded chlorophyll a/b binding protein in the albino leaves than in the green leaves. Our results suggest that, together with the concatenated negative pressure for subsequent photosynthetic processes, the albino phenotype is at least partly attributable to chloroplast inner membrane damage or to the impairment of photosynthetic pigment accumulation, which results from low Lhca1 expression.

Identification of gene fragments related to nitrogen deficiency in Eichhornia crassipes (Pontederiaceae).

Eichhornia crassipes is an aquatic plant native to the Amazon River Basin. It has become a serious weed in freshwater habitats in rivers, lakes and reservoirs both in tropical and warm temperate areas worldwide. Some research has stated that it can be used for water phytoremediation, due to its strong assimilation of nitrogen and phosphorus, and the accumulation of heavy metals, and its growth and spread may play an important role in environmental ecology. In order to explore the molecular mechanism of E. crassipes to responses to nitrogen deficiency, we constructed forward and reversed subtracted cDNA libraries for E. crassipes roots under nitrogen deficient condition using a suppressive subtractive hybridization (SSH) method. The forward subtraction included 2,100 clones, and the reversed included 2,650 clones. One thousand clones were randomly selected from each library for sequencing. About 737 (527 unigenes) clones from the forward library and 757 (483 unigenes) clones from the reversed library were informative. Sequence BlastX analysis showed that there were more transporters and adenosylhomocysteinase-like proteins in E. crassipes cultured in nitrogen deficient medium; while, those cultured in nitrogen replete medium had more proteins such as UBR4-like e3 ubiquitin-protein ligase and fasciclin-like arabinogalactan protein 8-like, as well as more cytoskeletal proteins, including actin and tubulin. Cluster of Orthologous Group (COG) analysis also demonstrated that in the forward library, the most ESTs were involved in coenzyme transportation and metabolism. In the reversed library, cytoskeletal ESTs were the most abundant. Gene Ontology (GO) analysis categories demonstrated that unigenes involved in binding, cellular process and electron carrier were the most differentially expressed unigenes between the forward and reversed libraries. All these results suggest that E. crassipes can respond to different nitrogen status by efficiently regulating and controlling some transporter gene expressions, certain metabolism processes, specific signal transduction pathways and cytoskeletal construction.

Profilin-2 increased expression and its altered interaction with β-actin in the striatum of 3-nitropropionic acid-induced Huntington’s disease in rats

Subacute systemic treatment with 3-nitropropionic acid (3-NP) causes specific lesions in the cortex and the striatum, and Huntington’s disease behavioral phenotypes in rats. We investigated differentially expressed genes in the striatum, and examined status of a highly expressed huntingtin interacting protein, profilin 2 (Pfn2) in relation to 3-NP-induced striatal neurodegeneration, employing both in vivo animal model and in vitro primary striatal neuronal cultures. Golgi staining of 3-NP-treated rat brain revealed significantly altered dendritic spine morphology and decreased spine density in the cortex and the striatum, as compared to the control. We employed suppression subtractive hybridization (SSH) method to screen differentially expressed genes during striatal neurodegeneration in these animals. Forward and reverse SSH provided a library of 188 clones, which were used for reverse northern dot blot analysis to identify greatly altered striatal-specific genes. Sequence analysis of the clones identified 23 genes, expressions of which were ⩾1.5-fold changed (16 up-regulated) in the striatum of 3-NP-treated rats. Immunoprecipitation assay showed decreased binding of Pfn2 with β-actin, the level of which remained unaffected in the striata and cortices of 3-NP-treated rats. Primary cultures of striatal glutamic acid decarboxylase-65/67 immunopositive GABAergic neurons revealed loss of co-existence of Pfn2 and β-actin in fluorescence imaging studies following 3-NP treatment for 24h. Since Pfn2 is known to regulate dendritic spine dynamics by interacting with β-actin, the reduction in its binding affinity to Pfn2 following 3-NP neurotoxic insult, and the accompanying aberrations of the dendritic spine structure and loss of spine density in striatal neurons suggest that Pfn2 may be involved in neurodegeneration in 3-NP-treated rat model of HD.

Cloning, expression, and purification of a new antimicrobial peptide gene from Musca domestica larva

Musca domestica (Diptera: Muscidae), the housefly, exhibits unique immune defences and can produce antimicrobial peptides upon stimulation with bacteria. Based on the cDNA library constructed using the suppression subtractive hybridization (SSH) method, a 198-bp antimicrobial peptide gene, which we named MDAP-2, was amplified by rapid amplification of cDNA ends (RACE) from M. domestica larvae stimulated with Salmonella pullorum (Enterobacteriaceae: Salmonella). In the present study, the full-length MDAP-2 gene was cloned and inserted into a His-tagged Escherichia coli prokaryotic expression system to enable production of the recombinant peptide. The recombinant MDAP-2 peptide was purified using Ni-NTA HisTrap FF crude column chromatography. The bacteriostatic activity of the recombinant purified MDAP-2 protein was assessed. The results indicated that MDAP-2 had in vitro antibacterial activity against all of the tested Gram− bacteria from clinical isolates, including E. coli (Enterobacteriaceae: Escherichia), one strain of S. pullorum (Enterobacteriaceae: Salmonella), and one strain of Pasteurella multocida. DNA sequencing and BLAST analysis showed that the MDAP-2 antimicrobial peptide gene was not homologous to any other antimicrobial peptide genes in GenBank. The antibacterial mechanisms of the newly discovered MDAP-2 peptide warrant further study.