Subtractive Hybridization Procedure Research Articles

Identification of Haemaphysalis longicornis Genes Differentially Expressed in Response to Babesia microti Infection.

Haemaphysalis longicornis is a tick and a vector of various pathogens, including the human pathogenetic Babesia microti. The objective of this study was to identify female H. longicornis genes differentially expressed in response to infection with B. microti Gray strain by using a suppression subtractive hybridization (SSH) procedure. A total of 302 randomly selected clones were sequenced and analyzed in the forward subtracted SSH cDNA library related to Babesia infection, and 110 clones in the reverse cDNA library. Gene ontology assignments and sequence analyses of tick sequences in the forward cDNA library showed that 14 genes were related to response to stimulus or/and immune system process, and 7 genes had the higher number of standardized sequences per kilobase (SPK). Subsequent real-time PCR detection showed that eight genes including those encoding for Obg-like ATPase 1 (ola1), Calreticulin (crt), vitellogenin 1 (Vg1) and Vg2 were up-regulated in fed ticks. Compared to uninfected ticks, infected ticks had six up-regulated genes, including ola1, crt and Vg2. Functional analysis of up-regulated genes in fed or Babesia-infected ticks by RNA interference showed that knockdown of crt and Vg2 in infected ticks and knockdown of ola1 in uninfected ticks accelerated engorgement. In contrast, Vg1 knockdown in infected ticks had delayed engorgement. Knockdown of crt and Vg1 in infected ticks decreased engorged female weight. Vg2 knockdown reduced B. microti infection levels by 51% when compared with controls. The results reported here increase our understanding of roles of H. longicornis genes in blood feeding and B. microti infection.

Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes

Global transcriptomic analysis via RNA-seq is often hampered by the high abundance of ribosomal (r)RNA in bacterial cells. To remove rRNA and enrich coding sequences, subtractive hybridization procedures have become the approach of choice prior to RNA-seq, with their efficiency varying in a manner dependent on sample type and composition. Yet, despite an increasing number of RNA-seq studies, comparative evaluation of bacterial rRNA depletion methods has remained limited. Moreover, no such study has utilized RNA derived from bacterial biofilms, which have potentially higher rRNA:mRNA ratios and higher rRNA carryover during RNA-seq analysis. Presently, we evaluated the efficiency of three subtractive hybridization-based kits in depleting rRNA from samples derived from biofilm, as well as planktonic cells of the opportunistic human pathogen Pseudomonas aeruginosa. Our results indicated different rRNA removal efficiency for the three procedures, with the Ribo-Zero kit yielding the highest degree of rRNA depletion, which translated into enhanced enrichment of non-rRNA transcripts and increased depth of RNA-seq coverage. The results indicated that, in addition to improving RNA-seq sensitivity, efficient rRNA removal enhanced detection of low abundance transcripts via qPCR. Finally, we demonstrate that the Ribo-Zero kit also exhibited the highest efficiency when P. aeruginosa/Staphylococcus aureus co-culture RNA samples were tested.

The Trametes hirsuta 072 laccase multigene family: Genes identification and transcriptional analysis under copper ions induction.

Laccases, blue copper-containing oxidases, ≿ an play an important role in a variety of natural processes. The majority of fungal laccases are encoded by multigene families that express closely related proteins with distinct functions. Currently, only the properties of major gene products of the fungal laccase families have been described. Our study is focused on identification and characterization of laccase genes, which are transcribed in basidiomycete Trametes hirsuta 072, an efficient lignin degrader, in a liquid medium, both without and with induction of laccase transcription by copper ions. We carried out production of cDNA libraries from total fungal RNA, followed by suppression subtractive hybridization and mirror orientation selection procedures, and then used Next Generation Sequencing to identify low abundance and differentially expressed laccase transcripts. This approach resulted in description of five laccase genes of the fungal family, which, according to the phylogenetic analysis, belong to distinct clusters within the Trametes genus. Further analysis established similarity of physical, chemical, and catalytic properties between laccases inside each cluster. Structural modeling suggested importance of the sequence differences in the clusters for laccase substrate specificity and catalytic efficiency. The implications of the laccase variations for the fungal physiology are discussed.

Specific Expression of a Novel Nodulin GmN479 Gene in the Infected Cells of Soybean (Glycine max) Nodules

Abstract A novel nodulin gene, GmN479 genomic clone composing of 3 630 nucleotides was isolated from mature soybean nodules using GmN479 cDNA as a probe by subtractive hybridization procedure. GmN479 encodes 170 amino acids with 2.09 kb nucleotides promoter region, and contains two important upstream promoter elements, one is a conserved “s-acting sequence motif 5′-AAAGAT-3′ controlling nodulin gene expression, and the other is typical CAAT boxes. GmN479 gene has a single zmc-fmger C 2H 2 domain YS CAF CQRGFSNAQALLGG HMNI H and a conserved motif, QALGGHMN m the zinc-finger with a short leucine repeat in the LDLELRLGL motif closed to C-terminal. These two conserved motifs share respectively higher identity with those in the floral regulator SUPERMAN gene, indicating that GmN479 may function as a transcriptional regulator, and is a likely candidate for playing a role in nodule-morphogenesis. Blotting data showed that GmN479 is a single copy presenting in the genome of soybean nodule, and its expression profile is similar to that of Lb-a, but it is different from that of ENOD2. GUS staining showed that GmN479 promoter just functions in the infected cells of nodules, indicating that the GmN479 is one of the truly symbiotically induced host genes, and belongs to a late nodulin gene. The expression pattern of GmN479 gene seems to imply that it may be closely related to the development of the nodule. In a sense, it may be a useful marker for identifying the development of the infected cell system in the nodules of soybean.

Genes encoding novel secreted and transmembrane proteins are temporally and spatially regulated during Drosophila melanogasterembryogenesis

BackgroundMorphogenetic events that shape the Drosophila melanogaster embryo are tightly controlled by a genetic program in which specific sets of genes are up-regulated. We used a suppressive subtractive hybridization procedure to identify a group of developmentally regulated genes during early stages of D. melanogaster embryogenesis. We studied the spatiotemporal activity of these genes in five different intervals covering 12 stages of embryogenesis.ResultsMicroarrays were constructed to confirm induction of expression and to determine the temporal profile of isolated subtracted cDNAs during embryo development. We identified a set of 118 genes whose expression levels increased significantly in at least one developmental interval compared with a reference interval. Of these genes, 53% had a phenotype and/or molecular function reported in the literature, whereas 47% were essentially uncharacterized. Clustering analysis revealed demarcated transcript groups with maximum gene activity at distinct developmental intervals. In situ hybridization assays were carried out on 23 uncharacterized genes, 15 of which proved to have spatiotemporally restricted expression patterns. Among these 15 uncharacterized genes, 13 were found to encode putative secreted and transmembrane proteins. For three of them we validated our protein sequence predictions by expressing their cDNAs in Drosophila S2R+ cells and analyzed the subcellular distribution of recombinant proteins. We then focused on the functional characterization of the gene CG6234. Inhibition of CG6234 by RNA interference resulted in morphological defects in embryos, suggesting the involvement of this gene in germ band retraction.ConclusionOur data have yielded a list of developmentally regulated D. melanogaster genes and their expression profiles during embryogenesis and provide new information on the spatiotemporal expression patterns of several uncharacterized genes. In particular, we recovered a substantial number of unknown genes encoding putative secreted and transmembrane proteins, suggesting new components of signaling pathways that might be incorporated within the existing regulatory networks controlling D. melanogaster embryogenesis. These genes are also good candidates for additional targeted functional analyses similar to those we conducted for CG6234.See related minireview by Vichas and Zallen:

Isolation and characterization of 11 microsatellite loci from Oropsylla hirsuta, a vector of sylvatic plague

The flea (Oropsylla hirsuta) is an important vector of the plague bacterium, Yersinia pestis, in black-tailed prairie dog (Cynomys ludovicianus) colonies. We developed 11 anonymous microsatellite primers for O. hirsuta using a subtractive hybridization procedure. All primers were polymorphic exhibiting 4-12 alleles.

Selective capture of transcribed sequences (SCOTS) of Actinobacillus pleuropneumoniae in the chronic stage of disease reveals an HlyX-regulated autotransporter protein

Actinobacillus pleuropneumoniae, an important respiratory pathogen in swine, is able to persist in host tissues for extended periods of time. In the study presented here, selective capture of transcribed sequences (SCOTS) analysis was used to identify genes expressed by A. pleuropneumoniae in the chronic stage of the disease (21 days post infection). After isolation and reverse transcription of RNA from infected lungs as well as from culture-grown A. pleuropneumoniae, transcribed A. pleuropneumoniae sequences were captured from infected lung tissue and subjected to a subtractive hybridization procedure of lung-derived against culture-derived A. pleuropneumoniae cDNA. Twenty-nine of the thirty-six genes that were identified as in vivo-expressed are involved in transport or metabolic processes. We identified a surface-associated putative 104 kDa subtilisin-like autotransporter serine protease, designated AasP, which has not been described in A. pleuropneumoniae to date. The gene was shown to be present in all 15 A. pleuropneumoniae serotypes. It is transcribed in porcine lung tissue on days 7 and 21 post infection. Under anaerobic conditions in vitro, its expression depends on the global anaerobic regulator HlyX. To our knowledge, this is the first report of an autotransporter protein being regulated by a global anaerobic regulator.

Subtractive hybridization for differential gene expression in mechanically unloaded rat heart

The objective of this study was to identify differentially expressed genes in the mechanically unloaded rat heart by suppression subtractive hybridization. In male Wistar-Kyoto rats, mechanical unloading was achieved by infrarenal heterotopic heart transplantation. Differentially expressed genes were investigated systematically by suppression subtractive hybridization. Selected targets were validated by Northern blot analysis, real-time RT-PCR, and immunoblot analysis. Maximal ADP-stimulated oxygen consumption (state 3) was measured in isolated mitochondria. Transplantation caused atrophy (heart-to-body weight ratio: 1.6 +/- 0.1 vs. 2.4 +/- 0.1, P < 0.001). We selected 1,880 clones from the subtractive hybridization procedure (940 forward and 940 reverse runs assessing up- or downregulation). The first screen verified 465 forward and 140 reverse clones, and the second screen verified 67 forward and 30 reverse clones. On sequencing of 24 forward and 23 reverse clones, 9 forward and 14 reverse homologies to known genes were found. Specifically, we identified reduced mRNA expression of complex I (-49%, P < 0.05) and complex II (-61%, P < 0.001) of the respiratory chain. Significant reductions were also observed on the respiratory chain protein level: -42% for complex I (P < 0.01), -57% for complex II (P < 0.05), and -65% for complex IV (P < 0.05). Consistent with changes in gene and protein expression, state 3 respiration was significantly decreased in isolated mitochondria of atrophied hearts, with glutamate and succinate as substrates: 85 +/- 27 vs. 224 +/- 32 natoms O.min(-1).mg(-1) with glutamate (P < 0.01) and 59 +/- 18 vs. 154 +/- 30 natoms O.min(-1).mg(-1) with succinate (P < 0.05). Subtractive hybridization indicates major changes in overall gene expression by mechanical unloading and specifically identified downregulation of respiratory chain genes. This observation is functionally relevant and provides a mechanism for the regulation of respiratory capacity in response to chronic mechanical unloading.

Isolation and identification of up-regulated genes in bermudagrass roots ( Cynodon dactylon L.) grown under petroleum hydrocarbon stress

The presence of petroleum hydrocarbons in soils is a drawback for plant growth. Recently, efforts to understand the toxicity of petroleum to plants have been oriented towards phytoremediation, since the main condition for successful phytoremediation of a petroleum-polluted soil is that plants grow under the stress imposed by the organic pollutant. However, there is not much information on the molecular mechanisms underlying the adaptation to this stress. In this work, we analyzed the mRNA expression profile of a plant used for phytoremediation, bermudagrass ( Cynodon dactylon), grown under petroleum stress. The calculated EC 50 toxic concentration of petroleum was 5.5 ± 1.1% W maya crude oil/ W soil ( W: weight). cDNA from roots of bermudagrass plants growing on soils containing 10% W maya crude oil/ W soil was used as tester against a control driver in suppression subtractive hybridization procedures. In this way, we isolated 24 unique cDNAs that are consistently over-expressed in roots of bermudagrass growing under petroleum stress. Comparative analysis of these cDNA sequences allowed us to classify them into six categories: general metabolism (e.g. fatty acids ligase, alcohol dehydrogenase, profilin, glycosyl transferases), signal transduction (e.g. G-protein, serine/threonine phosphatase, histidin kinases), nuclear transport (La-domain protein and nucleoporin 98), protein synthesis and degradation (ribosomal proteins, ubiquitin), unassigned biological activity (e.g. B12D) and sequences without identity. A theoretical analysis of the functions of these proteins in roots growing under petroleum hydrocarbon stress is also presented.

Identification of growth phenotype-related genes in Aspergillus oryzae by heterologous macroarray and suppression subtractive hybridization

Aspergillus oryzae requires polarized growth for colonization of solid substrates, and this growth phenotype differs from that seen in liquid medium. Various experimental approaches were used to identify genes that are differentially expressed when A. oryzae is grown on wheat kernels and in a wheat-based liquid medium. Hybridization of A. oryzae RNAs to a macroarray bearing cDNAs isolated from a library representing at least 16% of the total number of A. niger genes identified 14 differentially expressed cDNA clones, showing that heterologous macroarray analysis with an A. niger cDNA library can be used to identify regulated gene transcripts in the related species A. oryzae. Moreover, Northern analysis with a selection of eight probes for A. niger genes encoding proteins involved in morphological development and cell wall biosynthesis identified five more differentially expressed genes. A suppression subtractive hybridization procedure revealed another 12 differentially expressed genes. The results presented show that, of the 29 identified genes which are expressed at higher levels during growth on wheat kernels, six encode proteins that are functionally related to polarized growth, four encode products known to be involved in morphogenesis, three code for proteins related to cell wall composition, and nine of the cDNA clones encode novel proteins. These findings pinpoint genes associated with the changes in cellular morphogenesis seen in A. oryzae grown on wheat kernels as opposed to wheat-based liquid medium.

Differential Gene Expression in Response to Ventricular Unloading in Rat and Human Myocardium

Left ventricular unloading by mechanical assist devices induces myocardial atrophy. We aimed to systematically identify differentially expressed genes in a model of physiological atrophy (unloading of healthy rat myocardium) and compare these changes to those in a unloaded, failing human heart. Atrophy in rat hearts was induced by heterotopic transplantation of a donor heart into the abdomen of an isogenic recipient. After one week, donor and recipient RNA was isolated. Differential gene expression was assessed by subtractive hybridization. Two screens with radioactive probes were performed to verify differentially expressed clones. Positive clones were sequenced and cDNA of genes of known homology were used as probes for hybridization with RNA from separate atrophied rat hearts and human tissue from a normal, failing or failing and unloaded left ventricle. We picked 1880 clones from the subtractive hybridization procedure (940/940: forward/reverse runs assessing up- or down-regulation, respectively). The first screen verified 465/140 and the second screen verified 67/30 clones. 24/23 clones were sequenced and 14/10 homologies to known genes were found. In the atrophied heart, respiratory chain and metabolic genes were down-regulated (NADH-DH, cytochrome c oxidase, acetyl-CoA synthetase, myoglobin) and cellular recognition and stress genes were up-regulated (MHC1 and 2, HSP70). In the human heart, cytochrome c oxidase, acetyl-CoA synthetase, and myoglobin expression was increased in the failing heart and returned to normal with unloading. Unloading also resulted in up-regulation of HSP70. The genetic responses of failing human and healthy rat myocardium to mechanical unloading show similarities that appear to be independent of species differences and/or underlying disease. Thus, heterotopic heart transplantation is a relevant model for investigating the mechanisms of mechanical unloading.

Isolation of pigment cell specific genes in the sea urchin embryo by differential macroarray screening.

New secondary mesenchyme specific genes, expressed exclusively in pigment cells, were isolated from sea urchin embryos using a differential screening of a macroarray cDNA library. The comparison was performed between mRNA populations of embryos having an expansion of the endo-mesodermal territory and embryos blocked in secondary mesenchyme specification. To be able to isolate transcripts with a prevalence down to five copies per cell, a subtractive hybridization procedure was employed. About 400 putative positive clones were identified and sequenced from the 5' end. Gene expression analysis was carried out on a subset of 66 clones with real time quantitative PCR and 40 clones were positive. This group of clones contained sequences highly similar to: the transcription factor glial cells missing (gcm); the polyketide synthase gene cluster (pks-gc); three different members of the flavin-containing monooxygenase gene family (fmo); and a sulfotransferase gene (sult). Using whole mount in situ hybridization, it was shown that these genes are specifically expressed in pigment cells. A functional analysis of the S. purpuratus pks and of one S. purpuratus fmo was carried out using antisense technology and it was shown that their expression is necessary for the biosynthesis of the sea urchin pigment echinochrome. The results suggest that S. purpuratus pks, fmo and sult could belong to a differentiation gene battery of pigment cells.

Limitrin, a novel immunoglobulin superfamily protein localized to glia limitans formed by astrocyte endfeet.

We report the molecular cloning of a new member of the transmembrane-type immunoglobulin superfamily and designate the encoded protein as limitrin, since it localized selectively to glia limitans in mouse brain. Limitrin cDNA was obtained using a subtractive hybridization procedure designed to identify molecules responsible for blood-brain barrier function. Western blots using a limitrin-specific antibody demonstrated that the gene product is expressed significantly in mouse brain and primary murine astrocytes and is distributed in the plasma membrane. Immunohistochemical studies using confocal and electron microscopy clearly demonstrated highly polarized localization in astroglial endfeet in the perivascular region and under the pia mater in vivo. Limitrin is expressed in the spinal cord and in many areas of the brain, but not in the median eminence or subfornical organ (the circumventricular organs), where the blood-brain barrier is lacking. Disruption of the blood-brain barrier by cold injury resulted in a drastic reduction in limitrin expression. Furthermore, during retrieval from cold injury, the increased expression of limitrin in perivascular endfeet correlated with the recovery of angiogenesis in capillaries within the lesion margins. Our results suggest that limitrin is physically and functionally associated with the blood-brain barrier, implying that this protein may be useful as a diagnostic tool of barrier integrity.

Disease stress‐inducible genes of tobacco: expression profile of elicitor‐responsive genes isolated by subtractive hybridization

In order to investigate the change in mRNA profile during tobacco disease response, a subtractive hybridization procedure was used to generate a cDNA library for genes induced in tobacco (Nicotiana tabacum cv. Samsun NN) treated with oomycete elicitor. Database searches with the randomly isolated genes revealed that this cDNA library was enriched for reported disease stress‐responsive genes such as pathogenesis‐related proteins and cell wall protein genes. The expressions of eight newly isolated genes were induced by inoculation with the non‐pathogenic bacteria, Pseudomonas syringae pv. glycinea. The NtEIGs (N.tabacumelicitor‐inducible genes) showed similarity to genes for stellacyanin‐like protein (NtEIG‐A1), glutathione peroxidase (NtEIG‐C08), extensin‐like protein (NtEIG‐C29), WRKY transcription factor (NtEIG‐D48), glycine rich protein (NtEIG‐E17), β‐1, 3‐glucanase‐like protein (NtEIG‐E76), photoassimilate‐responsive protein‐1 (NtEIG‐E80) and wound‐induced protein (NtEIG‐D10). The expression patterns of NtEIGs in tobacco leaf in response to P. syringae pv. glycinea, salicylic acid (SA), methyl jasmonate (MeJA) and wound stress were analysed. The individual expression patterns of NtEIGs indicate that the transcriptional activation of NtEIGs is regulated by various signals and the products of NtEIGs are involved in different processes at different stages of the plant defence responses.

Identification and characterization of a Cryptococcus neoformans ATP binding cassette (ABC) transporter-encoding gene, CnAFR1, involved in the resistance to fluconazole.

Resistance to fluconazole is a possible event during prolonged suppressive drug therapy for cryptococ-cal meningitis, the most frequently encountered life-threatening manifestation of cryptococcosis. The knowledge of this resistance at the molecular level is important for management of cryptococcosis. In order to identify genes involved in azole resistance in Cryptococcus neoformans, a cDNA subtraction library technique was chosen as a strategy. First, a fluconazole-resistant mutant BPY22.17 was obtained from a susceptible clinical isolate BPY22 by in vitro exposure to the drug. Then, a subtractive hybridization procedure was used to compare gene expression between the obtained strains. We identified a cDNA overexpressed in the fluconazole-resistant strain BPY22.17 that was used as a probe to isolate the entire gene in a C. neoformans genomic library. Sequence analysis of this gene identified an ATP Binding Cassette (ABC) transporter-encoding gene called C. neoformans AntiFungal Resistance 1 (CnAFR1). Disruption of CnAFR1 gene in the resistant isolate (BPY22.17) resulted in an enhanced susceptibility of the knock-out mutant cnafr1 against fluconazole, whereas reintroduction of the gene in cnafr1 resulted in restoration of the resistance phenotype, thus confirming that CnAFR1 is involved in fluconazole resistance of C. neoformans. Our findings therefore reveal that an active drug efflux mechanism can be involved in the development of azole resistance in this important human pathogen.

Identification of genes induced by oxidized phospholipids in human aortic endothelial cells

Oxidized- l-α-1- Palmitoyl-2- Arachidonoyl- sn-glycero-3- Phosphoryl choline (Ox-PAPC), a component of mildly oxidized/minimally modified low-density lipoprotein (MM-LDL), accounts for many of the biological activities of MM-LDL. Having hypothesized that Ox-PAPC initiates gene expression changes in endothelial cells that result in enhanced endothelial/monocyte interactions and the subsequent development of atherosclerotic lesions, we used the suppression subtractive hybridization (SSH) procedure to compare mRNA isolated from PAPC-treated human aortic endothelial cells (HAEC) with mRNA isolated from Ox-PAPC-treated cells. Genes induced by Ox-PAPC but not by PAPC in HAEC included genes involved in signal transduction, extracellular matrix, growth factors, chemokines and several genes with unknown functions. The observed pattern of gene induction suggests that Ox-PAPC may play multiple roles in angiogenesis, atherosclerosis, and inflammation and wound healing.

Prestin, the Motor Protein of Outer Hair Cells

Prestin is a gene recently cloned from mammalian cochlear outer hair cells (OHC) using a single cell type, outer minus inner hair cell, specific suppressive subtractive hybridization procedure. The localization and gene expression profile of the prestin protein fits the pattern of OHC’s development of electromotility. When prestin is abundantly expressed in normally nonmotile kidney cells, nonlinear capacitance and motility that are normally only seen in OHCs can be recorded. Furthermore, both nonlinear capacitance and motility can be reduced by salicylate, a well-known inhibitor of electromotility. These data suggest that prestin is the motor protein of OHCs. Amino acid sequence and gene structure analysis indicate that prestin is the fifth member of a newly discovered anion transport family (SLC26) that includes PDS, DRA and DTDST, which are chloride-iodide transporters, Cl^–/HCO^–₃ exchangers or sulfate transporters. Prestin shares overall structure similarity with this anion transporter family. Recently, intracellular anions (chloride or bicarbonate) were found to be essential for OHC electromotility and prestin’s function.

Directed gap closure in large-scale sequencing projects.

A problem in many sequencing projects is the final closure of gaps left in the clone libraries, which serve as templates for sequencing, because of uncloned or unclonable genomic areas. By use of the Xylella fastidiosa genome as a test system, we present here an approach to generate, in a directed manner, sequence information from those gaps. We suggest using the complete clone library as a competitor against the genomic DNA of interest in a subtractive hybridization procedure similar to representational difference analysis (RDA). The resulting sequence information can be used to screen selectively other clone resources or serve directly for gap closure.

Mitogen-activated Protein Kinase Phosphatase 1 Activity Is Necessary for Oxidized Phospholipids to Induce Monocyte Chemotactic Activity in Human Aortic Endothelial Cells

Entrapment and oxidation of low density lipoproteins (LDL) in the sub-endothelial space is a key process in the initiation of atherosclerotic lesion development. Functional changes induced by oxidized lipids in endothelial cells are early events in the pathogenesis of atherosclerosis. Oxidized-l-alpha-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (ox-PAPC), a major component of minimally modified/oxidized-LDL (MM-LDL) mimics the biological activities assigned to MM-LDL both in vitro in a co-culture model as well as in vivo in mice. We hypothesized that ox-PAPC initiates gene expression changes in endothelial cells that result in enhanced endothelial/monocyte interactions. To analyze the gene expression changes that oxidized lipids induce in endothelial cells, we used a suppression subtractive hybridization procedure to compare mRNA from PAPC-treated human aortic endothelial cells (HAEC) with that of ox-PAPC-treated cells. We report here the identification of a gene, mitogen-activated protein kinase phosphatase 1 (MKP-1), that is rapidly and transiently induced in ox-PAPC-treated HAEC. Inhibition of MKP-1 using either the phosphatase inhibitor sodium orthovanadate or antisense oligonucleotides prevents the accumulation of monocyte chemotactic activity in ox-PAPC-treated HAEC supernatants. Furthermore, we show that decreased monocyte chemotactic activity in HAEC treated with sodium orthovanadate or MKP-1 antisense oligonucleotides is due to decreased MCP-1 protein. Our results implicate a direct role for MKP-1 in ox-PAPC-induced signaling pathways that result in the production of MCP-1 protein by ox-PAPC-treated HAEC.

8] Subtractive hybridization-based identification of genes uniquely expressed or hyperexpressed during biofilm growth

Publisher Summary This chapter discusses Biofilms that display several phenotypes that are fundamentally different from those of planktonically grown bacteria, including decreased susceptibility to antimicrobial agents, and host immune response. The biofilm represents a unique mode of growth distinct from planktonic bacteria. A subtractive hybridization procedure has been developed to identify genes uniquely expressed or hyperexpressed during the biofilm mode of growth. In its simplest form, subtractive hybridization involves hybridizing nucleic acid from an induced population of cells with excess nucleic acid from an uninduced population, with the goal of eliminating transcripts common to both populations, and thereby increasing the concentration of sequences unique to the tester population. Subtractive hybridization has a number of inherent features that distinguish it from traditional genetic approaches used to identify genes induced under specific growth conditions. Transposon mutagenesis would be effective only for identification of factors absolutely required for biofilm formation. Subtractive hybridization, however, has the potential for identifying all factors that are uniquely expressed or hyper-expressed by cells during the biofilm mode of growth in a single assay. Traditional subtractive hybridization procedures often require a large amount of starting material, and are inefficient for the identification of low-abundance transcripts because of prolonged hybridization and inefficient separation steps. The subtractive hybridization protocol is dependent on differential polymerase chain reaction (PCR), amplification of biofilm (tester), and planktonic (driver) populations of cDNA, resulting in population-specific biochemical properties. This enables selection against transcripts commonly expressed in both populations of cells, and allows for selection of transcripts unique to the tester population.