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Drought transcriptome analysis of finger millet (Eleusine coracana) by cDNA subtraction identified drought responsive genes that have a potential role in drought tolerance. Through virus-induced gene silencing (VIGS) in a related crop species, maize (Zea mays), several genes, including a G-BOX BINDING FACTOR 3 (GBF3) were identified as candidate drought stress response genes and the role of GBF3 in drought tolerance was studied in Arabidopsis thaliana. Overexpression of both EcGBF3 and AtGBF3 in A. thaliana resulted in improved tolerance to osmotic stress, salinity and drought stress in addition to conferring insensitivity to ABA. Conversely, loss of function of this gene increased the sensitivity of A. thaliana plants to drought stress. EcGBF3 transgenic A. thaliana results also suggest that drought tolerance of sensitive plants can be improved by transferring genes from far related crops like finger millet. Our results demonstrate the role of GBF3 in imparting drought tolerance in A. thaliana and indicate the conserved role of this gene in drought and other abiotic stress tolerance in several plant species.

The microsporidium Nosema ceranae is a high prevalent parasite of the European honey bee (Apis mellifera). This parasite is spreading across the world into its novel host. The developmental process, and some mechanisms of N. ceranae-infected honey bees, has been studied thoroughly, however, few studies have been carried out in the mechanism of gene expression in N. ceranae during the infection process. We therefore performed the suppressive subtractive hybridization (SSH) approach to investigate the candidate genes of N. ceranae during its infection process. All 96 clones of infected (forward) and non-infected (reverse) library were dipped onto the membrane for hybridization. A total of 112 differentially expressed sequence tags (ESTs) had been sequenced. For the host responses, 20% of ESTs (13 ESTs, 10 genes, and 1 non-coding RNA) from the forward library and 93.6% of ESTs (44 ESTs, 28 genes) from the reverse library were identified as differentially expressed genes (DEGs) of the hosts. A high percentage of DEGs involved in catalytic activity and metabolic processes revealed that the host gene expression change after N. ceranae infection might lead to an unbalance of physiological mechanism. Among the ESTs from the forward library, 75.4% ESTs (49 ESTs belonged to 24 genes) were identified as N. ceranae genes. Out of 24 N. ceranae genes, nine DEGs were subject to real-time quantitative reverse transcription PCR (real-time qRT-PCR) for validation. The results indicated that these genes were highly expressed during N. ceranae infection. Among nine N. ceranae genes, one N. ceranae gene (AAJ76_1600052943) showed the highest expression level after infection. These identified differentially expressed genes from this SSH could provide information about the pathological effects of N. ceranae. Validation of nine up-regulated N. ceranae genes reveal high potential for the detection of early nosemosis in the field and provide insight for further applications.

Transmembrane protein 132A (TMEM132A) was first isolated from rat brain using PCR-selected cDNA subtraction, and it was found to be predominantly expressed in the brain. However, the transcriptional regulation of the TMEM132A gene has not been fully characterized. In this study, we characterized the promoter activity of the 880-bp region upstream of the mouse TMEM132A, identifying several putative sites recognized by transcription factors, which are highly conserved between the mouse and human TMEM132A genes. Using four different mouse cell lines (Neuro2a, NSC-34, NIH3T3, and Raw264.7), we first evaluated the intrinsic levels of TMEM132A mRNA and protein expression. Interestingly, TMEM132A mRNA was expressed in all four cell lines, whereas the protein was negligible in Raw264.7 cells even by transfection of TMEM132A gene. Then, we analyzed the TMEM132A promoter activity using serial deleted constructs, finding it was nearly same pattern in all four cell lines. A mutational analysis of the TMEM132A promoter identified a critical region for its activation just upstream of the transcriptional start site. Finally, we investigated the levels of TMEM132A mRNA and protein after exposure to five different neurotoxic stimuli, including thapsigargin, tunicamycin, serum starvation, homocysteine, and hydrogen peroxide. Treatment with thapsigargin, a calcium modulating agent, markedly attenuated the levels of TMEM132A mRNA and protein in NSC-34 cells. These results give new insight into the mechanisms involved in regulating TMEM132A expression, and suggest that several transcriptional and post-transcriptional pathways regulate TMEM132A expression under developmental and pathophysiological conditions.

Some cultivars of cucumbers, melons, pumpkins, and zucchini, which are members of the Cucurbitaceae family, are uniquely subject to contamination by hydrophobic pollutants such as the organohalogen insecticides DDT. However, the molecular mechanisms for the accumulation of these pollutants in cucurbits have not been determined. Here, cDNA subtraction analysis of Cucurbita pepo cultivars that are low and high accumulators of hydrophobic contaminants revealed that a gene for zinc finger proteins (ZFPs) are preferentially expressed in high accumulators. The cloned CpZFP genes were classified into 2 types: (1) the PBG type, which were expressed in C. pepo cultivars Patty Green, Black Beauty, and Gold Rush, and (2) the BG type, which were expressed in Black Beauty and Gold Rush. Expression of these CpZFP genes in transgenic tobacco plants carrying an aryl hydrocarbon receptor-based inducible gene expression system significantly induced β-glucuronidase activity when the plants were treated with a polychlorinated biphenyl (PCB) compound, indicating that highly hydrophobic PCBs accumulated in the plants. In transgenic tobacco plants carrying CpZFPs, accumulation of dioxins and dioxin-like compounds increased in their aerial parts when they were cultivated in the dioxin-contaminated soil. In summary, we propose that addition of CpZFP genes is a promising tool for conferring noncucurbits with the ability to accumulate hydrophobic contaminants.

Low temperature is the main limiting factor for cultivation expansion, fast growth, and high yield of Eucalyptus species. To investigate the mechanism of their cold tolerance, a cDNA subtraction library representing the cold-induced genes of Eucalyptus dunnii was constructed using suppression subtractive hybridization (SSH) technique. A gene encoding a calmodulin-binding protein (CaMBP) was identified from the SSH library, and the expression pattern of CaMBP under cold stress was further evaluated through reverse transcription — quantitative polymerase chain reaction (RT-qPCR). The expressions of EguCBF1a and EdCaMBP increased already after 4 h of the cold stress, supporting the idea that the CaMBP gene may have a function in the survival of Eucalyptus during winter. A full-length mRNA sequence of 1 808 bp was obtained via a rapid amplification of cDNA ends method, and the sequence was subsequently deposited to GenBank (accession No. JX401571). The CaMBP cDNA of E. dunnii contained a single open reading frame of 1 362 bp, a 5t’ untranslated region of 175 bp, and a 3t’ untranslated region of 268 bp. Multiple sequence alignment and phylogenetic analysis indicated that CaMBP of E. dunnii shared 56 to 84 % identities with the CaMBPs of other plants and was similar to that of Ricinus communis, Medicago truncatula, and Gossypium hirsutum.

Jatropha curcas has been widely studied at the molecular level due to its potential as an alternative source of fuel. Many of the reports till date on this plant have focussed mainly on genes contributing to the accumulation of oil in its seeds. A suppression subtractive hybridization strategy was employed to identify genes which are differentially expressed in the mid maturation stage of J. curcas seeds. Random expressed sequence tag sequencing of the cDNA subtraction library resulted in 385 contigs and 1,428 singletons, with 591 expressed sequence tags mapping for enzymes having catalytic roles in various metabolic pathways. Differences in transcript levels in early and mid-to-late maturation stages of seeds were also investigated using sequence information obtained from the cDNA subtraction library. Seven out of 12 transcripts having putative roles in central carbon metabolism were up regulated in early seed maturation stage while lipid metabolism related transcripts were detected at higher levels in the later stage of seed maturation. Interestingly, 4 of the transcripts revealed putative alternative splice variants that were specifically present or up regulated in the early or late maturation stage of the seeds. Transcript expression patterns from the current study using maturing seeds of J. curcas reveal a subtle balancing of oil accumulation and utilization, which may be influenced by their energy requirements.

The preinfection stage of establishment of the mycorrhiza before physical contact between symbionts is crucial, as changes that occur throughout mycorrhiza formation are set in motion at this time. To improve the understanding of the molecular mechanisms involved in the fungus Pisolithus tinctorius during its interaction with its symbiotic partner Pinus massoniana, we set up a cellophane-separated culture system avoiding physical contact between the symbionts. A cDNA subtraction library representing the differentially expressed genes of P.tinctorius during the preinfection stage was constructed using suppression subtractive hybridization combined with mirror orientation selection (SSH/MOS). Differential screening of the SSH library allowed us to identify 133 unique cDNA clones. Sequences analysis of the expressed sequence tags (ESTs) showed that they represent 23 differentially expressed genes, of which 10 are involved in metabolism and energy, transcription and protein fate, membrane proteins and signaling, and cell rescue and defense, and 13 encoding hypothetical proteins with unknown function. Some fungal genes have not been previously identified in other ectomycorrhizal associations. Expression of the genes that code for ubiquitin-conjugating enzyme E2, 40S ribosomal protein, maternal g10 transcript, ADP ribosylation factor 6, and cytochrome P450 were evaluated by RT-qPCR, confirming the activation of these genes during the preinfection stage.

Fusarium is the most common flax pathogen causing serious plant diseases and in most cases leading to plant death. To protect itself, the plant activates a number of genes and metabolic pathways, both to counteract the effects of the pathogen, and to eliminate the threat. The identification of the plant genes which respond to infection is the approach, that has been used in this study. Forty-seven flax genes have been identified by means of cDNA subtraction method as those, which respond to pathogen infection. Subtracted genes were classified into several classes and the prevalence of the genes involved in the broad spectrum of antioxidants biosynthesis has been noticed. By means of semi-quantitative RT-PCR and metabolite profiling, the involvement of subtracted genes controlling phenylpropanoid pathway in flax upon infection was positively verified. We identified the key genes of the synthesis of these compounds. At the same time we determined the level of the metabolites produced in the phenylpropanoid pathway (flavonoids, phenolic acids) in early response to Fusarium attack by means of GC-MS technique. To the best of our knowledge this is the first report to describe genes and metabolites of early flax response to pathogens studied in a comprehensive way.

Sexually dimorphic neural circuits are essential for reproductive behaviour. The molecular basis of sexual dimorphism in the silkworm moth (Bombyx mori) brain, however, is unclear. We conducted cDNA subtraction screening and identified nervous wreck (Bmnwk), a synaptic growth regulatory gene, whose expression is higher in the male brain than in the female brain of the silkworm. Bmnwk was preferentially expressed in the brain at the late pupae and adult stages. In situ hybridization revealed that Bmnwk is highly expressed in the optic lobe of the male moth brain. These findings suggest that Bmnwk has a role in the development and/or maintenance of the optic lobe in the male silkworm brain.

The macroalga Ulva limnetica K. Ichihara et S. Shimada is the only known Ulva species to be distributed exclusively in freshwater and is restricted to freshwater bodies in the Ryuku archipelago. Molecular phylogenetic analysis suggests that U. limnetica originally evolved from marine forms of Ulva. The mechanisms of adaptation to freshwater in Ulva spp. are poorly understood. In this study, we isolated genes potentially involved in adaptation or tolerance to freshwater conditions in U. limnetica, using suppression subtractive hybridization between mRNAs of samples cultured in freshwater and seawater conditions. A total of 219 genes, up-regulated by the exposure of the macroalga to freshwater, were isolated. Reverse transcription–PCR (RT–PCR) revealed 39 clones, including malate dehydrogenase, soluble starch synthase, triosephosphate isomerase, plastid ribosomal protein, DnaJ-like protein, and chloroplast ascorbate peroxidase (APX), which were specifically or preferentially expressed in freshwater conditions. These 39 clones were also analyzed for their temporal transcriptional response to freshwater conditions. A large majority of these up-regulated genes showed a transient peak of expression after 1–4 h, followed in the next 24 h by a decrease to a stable level (over the 7 d of the experiment). After the initial response peak, the level of expression either remained higher than in the control (long-term response) or returned to a level similar to pretreatment level. A few genes showed a more delayed response (i.e., after several days) to freshwater exposure. Finally, we discussed the possible contributions of the freshwater-induced genes in the acquisition of freshwater adaptation or tolerance of U. limnetica.

The carposporophyte life-history phase is unique to florideophycean algae and its morphological development has been thoroughly investigated. Carposporophytes are tiny, generally embedded in female gametophyte tissues, and difficult to investigate via traditional molecular methods. However, subtractive cloning of differentially expressed genes from small amounts of tissue is now possible thanks to recent methodological advances. We applied a cDNA subtraction method using magnetic beads and PCR to isolate carposporophyte-specific cDNAs from Gracilariopsis andersonii (Gracilariales, Rhodophyta). Messenger RNAs extracted from carposporophyte tissues and vegetative thalli of the gametophyte were captured on oligo-dT magnetic beads, followed by first-strand cDNA synthesis in situ. Subtractive hybridization between the amplified second-strand carposporophyte cDNA in solution and the first-strand gametophyte cDNA attached to magnetic beads was conducted to remove common genes present in both gametophyte and carp...

Cotton boll development is the result of a genetically programmed process influenced by environmental factors. Sequence information derived from advanced EST sequencing is an essential resource for functional genomics studies. The present study was undertaken to understand the gene expression profile of the early boll development stage. PCR select cDNA subtraction was employed to obtain subtracted library using cDNA prepared from −5 Days Post Anthesis stage of bud as a tester and cDNA from leaves as a driver. Differential screening of 868 positive single inserts by cDNA macroarray resulted in 170 ESTs from −5 days post anthesis bud which were further assembled into 64 contigs. Obtained results were validated by quantitative PCR for selected genes. Functional annotation showed the presence of majority of genes involved in transport processes, stress, nucleotide binding and cytoskeleton. The role of carbohydrate metabolism in bud and role of tubulin6 in fibre cell initiation were discussed.

The hair follicle is one of the skin appendages that develops through reciprocal epithelial-mesenchymal interactions. Although a large number of studies have been made on the mechanisms of hair follicle development, the whole molecular mechanism that governs hair follicle development remains poorly defined. To further understand the molecular basis of hair follicle development, it is necessary to identify genes that drive hair morphogenesis. As an initial approach, we attempted to identify gene products associated with mouse hair follicle development. Genes upregulated in the vibrissal hair placodes were screened by polymerase chain reaction (PCR)-based cDNA subtraction. The genes thus isolated were evaluated for their hair development-associated induction and spatiotemporal expression by quantitative reverse-transcription-PCR analysis and whole-mount in situ hybridization, respectively. Finally, we identified four genes whose upregulation and spatiotemporal expression in developing hair follicles were confirmed. Successful identification of novel hair development-associated genes will be informative as clues for further characterization of hair follicle development at the molecular level.

To gain a better understanding of differentially expressed sequence tags (ESTs) for aluminum (Al) tolerance and to investigate the molecular mechanisms of Al toxicity, cDNA subtraction libraries were generated from Al-stressed roots of alfalfa (Medicago sativa L.) compared with no Al-stressed ones, employing suppression subtractive hybridization. Using differential screening technique in which the probes were labeled with DIG, we identified 45 non-redundant ESTs in Al-stressed alfalfa root tips with significantly altered expression. Among the up-regulated ESTs, we have found genes encoding identified proteins, including malate dehydrogenase, 6-phosphogluconate dehydrogenase, peroxidase, and an ABC transporter, while the down-regulate genes included ATPase, secretory carrier membrane protein 2, pectinesterase inhibitor. In addition, two novel ESTs, EW678752 and EY976957, up- and down-regulated by Al stress were sequenced. Analyzed by real-time PCR, the expressions of EST EW678718, EW678739, EY976969 and EW678728, which encode for ABC transporter, malate dehydrogenase, peroxidase and 6-phosphogluconate dehydrogenase correspondingly, increased 1.64-, 2.75-, 3.27- and 6.54-folds, respectively, and the expression of EY976957 encoding for ATPase decreased 3.27 folds. The expression of EST EW678752 increased 34.54-fold, while that of EY976957 decreased 16.68 folds. It suggested that the two novel ESTs maybe play a significant role in the aluminum tolerance of alfalfa.

A novel gene (BmK5) expressed in the central nervous system of the silkworm, Bombyx mori, was isolated using a cDNA subtraction method. BmK5 was first cloned as a candidate regulator of diapause hormone release from subesophageal ganglion via corpus cardiacum-corpus allatum into the hemolymph; however, subsequent analyses revealed that the gene expression patterns in brain-subesophageal ganglion complexes did not differ between diapause and nondiapause egg producers. The deduced amino acid sequence showed the characteristics of secretory protein precursor or nuclear localization protein. Immunohistochemical experiments with an anti-BmK5 antibody revealed that BmK5 precursor protein exists in the cytoplasm of specific cells of brain and frontal ganglion, but not in the nuclei. In addition, a peptide (GSGTKVGGAGAATKVVTKSGS-NH(2)) possibly processed from the BmK5 precursor protein was immunohistochemically detected in the axons connecting the anti-BmK5 antibody-positive cells to the neurohemal organ, corpus cardiacum-corpus allatum. These results suggest that BmK5 encodes a precursor of the novel neurosecretory protein and that several mature peptides are released into the hemolymph via the corpus cardiacum-corpus allatum, although the functions of these peptides are yet unclear.

The grapevine rootstock cultivar 'Borner' is resistant against phylloxera (Daktulosphaira vitifoliae Fitch), one of the major pests in viticulture. In contrast to tolerant or sensitive rootstocks, 'Borner' roots react to a phylloxera attack with a hypersensitive response exhibiting features of programmed cell death. Using cDNA subtraction in 'Borner' roots induced by IAA (a cecidogenic component of the Phylloxera saliva) and analysed at different times post induction, differential expression of several genes putatively involved in phylloxera resistance was established. Furthermore, DNA microarray analysis of subtracted and unsubtracted cDNA populations using a heterologous system (Arabidopsis thaliana) yielded more than 600 genes differentially expressed at various times after induction. Recently, a quantitative analysis of differentially expressed genes was initiated using an advanced microarray techniques ('Geniom one', febit AG) with oligo-DNA custom chips harbouring more than 450 relevant entries of the Vitis UniGen databank and a number of pathogen response-related genes of A. thaliana. Using this method, 25 up-and 7 down-regulated genes could be identified in 'Borner'. Differential expression of several of these candidate genes has been validated by quantitative RT-PCR.

Maize (Zea mays L.) is an important crop that is poorly adapted to cold stress; very little is known about the components of the response to stress. By means of the cDNA subtraction technique, several novel genes, responsive to cold stress, have been identified, including ZmCOI6.1, the function of which is unknown. The predicted ZmCOI6.1 amino acid sequence and its homologue are very similar to proteins in rice and Arabidopsis, suggesting that it belongs to a conserved group of plant proteins. Analysis of the ZmCOI6.1 promoter sequence revealed several conserved stress-responsive cis-acting elements. Further characterization of expression showed that ZmCOI6.1 was induced not only by cold, but also by drought and salinity as well as by the signaling molecules abscisic acid and salicylic acid, indicating that ZmCOI6.1 is a stress-responsive gene. ZmCOI6.1 is alternatively spliced and yields two transcripts, the level of which change depending on the stress, indicating a possible mechanism of regulation at the splicing level. Constitutive expression of ZmCOI6.1 in Arabidopsis yielded plants that were less tolerant to abiotic stress, providing evidence that ZmCOI6.1 may be a negative regulator.

In a search for differentially expressed genes in peel pitting of ‘Navel’ orange fruit (Citrus sinensis L. Osbeck), a cDNA subtraction library was constructed and a sequence encoding expansin-like gene was isolated and identified as pitting related gene. Based on sequence information derived from this fragment, a full-length cDNA (CsEXP, GenBank accession no. FJ769424) of 1,083 nucleotides encoding expansin was isolated from ‘Navel’ orange by RACE approaches. CsEXP encoded a protein of 254 amino acid residues with an open reading frame located in the region between 52 and 816 bp. The calculated molecular weight of the mature protein was 27.05 kDa and theoretical isoelectric point was 7.93. The deduced protein contained conserved domains of expansin: the histidine-phenylalanine-aspartate motif in central portion, cysteine residues in N-terminus, and tryptophan residues in C-terminal region. The expression of CsEXP was higher in pitting than the control. Exposure of fruit to stresses, including wounding, anoxia, low temperature (4°C), and treatment with ethylene, increased CsEXP mRNA levels in comparison with the control untreated fruit, whereas high temperature (40°C) decreased its mRNA levels. Since low temperature, low oxygen and wounding were suspected factors inducing peel pitting of citrus fruit. The present results provided us a clue that CsEXP may play a role in response to peel pitting related stresses.

The cultivar ‘Borner’ is one of the Phylloxera resistant rootstocks used in Europe. In contrast to tolerant or sensitive rootstocks, ‘Borner’ roots react to a Phylloxera attack by a cell necrosis around the puncture. Histological studies sug-gested that the reaction is a kind of hypersensitive response. The aim of this study was to identify genes differentially expressed during the HR. HR was chemically induced in root cells by the application of IES. After a cDNA subtraction of induced and non-induced material, the subtracted cDNA-samples were hybridized to Arabidopsis microarray chips to identify differentially expressed candidate genes. The microarray data were analysed and differentially expressed genes were grouped into different functional categories, e.g., signal transduction pathways, transcription factors, defence associated genes. Primers were designed to target genes of interest putatively involved in the HR. So far 12 ESTs of ‘Borner’ differentially regulated in roots undergoing a HR have been sequenced and annotated. INTRODUCTION

Malassezia species are associated with pityriasis versicolor (PV) and its depigmented variant pityriasis versicolor alba (PVa), widespread fungal skin infections in humans. The pathogenesis of PV and PVa remains unclear, including their clinical and histological symptoms such as hyper- and depigmentation, reduced responsiveness to ultraviolet radiation and lack of inflammatory reaction despite high fungal load. Pigments produced by M. furfur are possibly involved in the pathogenesis of PV. In vitro, M. furfur produces a wide range of pigments and fluorochromes when cultured with tryptophan as the sole nitrogen source. We have begun to analyse the molecular basis of pigment production by searching for genes associated with tryptophan-based pigment production. A suppression subtractive hybridization (SSH) protocol was used to identify genes expressed in M. furfur cells producing pigments, but not in non-induced cells. SSH was performed 3 and 5 h after onset of pigment induction. Up-regulation of genes in the pigment-producing cells was confirmed by reverse northern analysis. More than 1,500 cDNA sequences of both the indicated time points were analysed. We identified a wide variety of genes associated with metabolism and several genes with unknown function are specifically expressed during pigment production. Furthermore, a fraction of genes possibly involved in different steps of the newly discovered indolic pathway of M. furfur were expressed in pigment producing cells. These data provide the first molecular insight into pigment production of M. furfur.

Nariva virus (NarPV) was isolated from forest rodents (Zygodontomys b. brevicauda) in eastern Trinidad in the early 1960s. Initial classification within the family Paramyxoviridae was based mainly on morphological observations including the structure of nucleocapsids and virion surface projections. Here, we report the characterization of the complete genome sequence of NarPV. The genome is 15,276 nucleotides in length, conforming to the rule-of-six, and has a genome organization typical of most members of the family, with six transcriptional units in the order 3'-N-P-M-F-H-L-5'. The gene junctions contain highly conserved gene start and stop signals and a tri-nucleotide intergenic sequence present in most members of the subfamily Paramyxovirinae. Sequence comparison studies indicate that NarPV is most closely related to Mossman virus, which was isolated from wild rats (Rattus leucopus) in Queensland, Australia, in 1970. This study confirmed the classification of NarPV as a member of the subfamily Paramyxovirinae and established the close genome organization and sequence relationship between the two rodent paramyxoviruses isolated almost a decade apart and from two locations separated by more than 15,000 km.

Maternal transcripts are indispensable and play an essential role in regulation of the resumption of meiosis, nuclear reprogramming and subsequent development. In this study, we conducted interactive subtraction analysis to investigate the dynamic changes in the gene expression profiles of mouse oocytes after artificial activation. To accomplish this, we compared the cDNA libraries of freshly ovulated metaphase II (MII) oocytes and pronuclear-stage eggs at 8 h after activation, which were obtained by interactive subtraction. After constructing the cDNA libraries, we sequenced 635 subtracted clones of differentially expressed genes and the identities of 515 of these genes were determined by online BLAST analysis. The analysis revealed that genes down-regulated after activation were mainly responsible for enzyme and receptor activities, and also for DNA, chromatin, ATP, protein, and ion binding. On the other hand, genes up-regulated after activation were mainly responsible for enzyme activities, io...

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.

During development, cloned embryos often undergo embryonic arrest at any stage of embryogenesis, leading to diverse morphological abnormalities. The long-term effects resulting from embryo cloning procedures would manifest after birth as early death, obesity, various functional disorders, and so forth. Despite extensive studies, the parameters affecting the developmental features of cloned embryos remain unclear. The present study carried out extensive gene expression analysis to screen a cluster of genes aberrantly expressed in embryonic stem cell-cloned blastocysts. Differential screening of cDNA subtraction libraries revealed 224 differentially expressed genes in the cloned blastocysts: eighty-five were identified by the BLAST search as known genes performing a wide range of functions. To confirm their differential expression, quantitative gene expression analyses were performed by real-time PCR using single blastocysts. The genes Skp1a, Canx, Ctsd, Timd2, and Psmc6 were significantly up-regulated, whereas Aqp3, Ak3l1, Rhot1, Sf3b3, Nid1, mt-Rnr2, mt-Nd1, mt-Cytb, and mt-Co2 were significantly down-regulated in the majority of embryonic stem cell-cloned embryos. Our results suggest that an extraordinarily high frequency of multiple functional disorders caused by the aberrant expression of various genes in the blastocyst stage is involved in developmental arrest and various other disorders in cloned embryos.

cDNAs specifically expressed at the basidiome stage were isolated by using PCR-selected cDNA subtraction in order to study gene regulation during porous-hymenium basidiomatal formation in Antrodia cinnamomea. blastx results suggested that most of the expressed sequence tags (52.4-69.5%) had no significant protein homology to genes from other published living things. cDNAs particularly expressed at different growing conditions were identified using cDNA microarray analysis. Reverse transcriptase PCR analyses confirmed that the clone putative to P-type ATPase, various cytochrome P450s and some unknown genes were abundant at natural basidiomes while endoglucanase was abundant at the tissue from artificial medium.

Molecular biology tools can be used to monitor and optimize biological treatment systems, but the application of nucleic acid-based tools has been hindered by the lack of available sequences for environmentally relevant biodegradation genes. The objective of our work was to extend an existing molecular method for eukaryotes to prokaryotes, allowing us to rapidly identify differentially expressed genes for subsequent sequencing. Suppression subtractive hybridization (SSH) PCR cDNA subtraction is a technique that can be used to identify genes that are expressed under specific conditions (e.g., growth on a given pollutant). While excellent methods for eukaryotic SSH PCR cDNA subtraction are available, to our knowledge, no methods previously existed for prokaryotes. This work describes our methodology for prokaryotic SSH PCR cDNA subtraction, which we validated using a model system: Pseudomonas putida mt-2 degrading toluene. cDNA from P. putida mt-2 grown on toluene (model pollutant) or acetate (control substrate) was subjected to our prokaryotic SSH PCR cDNA subtraction protocol to generate subtraction clone libraries. Over 90% of the sequenced clones contained gene fragments encoding toluene-related enzymes, and 20 distinct toluene-related genes from three key operons were sequenced. Based on these results, prokaryotic SSH PCR cDNA subtraction shows promise as a targeted method for gene identification.

Homeotic transformation of stamens into pistil-like structures (called pistillody) has been reported in cytoplasmic substitution (alloplasmic) lines of bread wheat (Triticum aestivum) having the cytoplasm of a wild relative species, Aegilops crassa. Our previous studies indicated that pistillody is caused by alterations of the class B MADS-box gene expression pattern associated with mitochondrial gene(s) in the Ae. crassa cytoplasm. To elucidate the nuclear gene involved in the cross-talk between pistillody-related mitochondrial gene(s) and nuclear homeotic genes, we performed cDNA subtraction analysis using cDNAs derived from young spikes of a pistillody line and a normal line. As a result, we identified a protein kinase gene, WPPK1 (wheat pistillody-related protein kinase 1), which is upregulated in the young spikes of the pistillody line. RT-PCR analysis indicated that WPPK1 is strongly expressed in pistils and pistil-like stamens in the pistillody line, suggesting that it is involved in the formation of pistil-like stamens as well as pistils. The full-length cDNA sequence for WPPK1 showed high similarity with a flowering plant PVPK-1 protein kinase, and phylogenetic analysis indicated that it is a member of AGC group protein kinases. Furthermore, a phosphorylation assay indicated that it has protein kinase activity. In situ hybridization analysis revealed that WPPK1 is expressed in developing pistils and pistil-like stamens as well as in their primordia. These indicate that in the alloplasmic line, WPPK1 plays a role in formation and development of pistil-like stamens.

The plant regulator methyl-jasmonate (MeJA) mediates diverse developmental processes and defense responses which induce a variety of secondary metabolites. This study reports the regulatory network of MeJA-induced genes in adventitious root cultures of the medicinal herb Bupleurum kaoi. A cDNA microarray containing 465 unique expressed sequence tags (ESTs) as probe sets, generated with a PCR-Select cDNA Subtraction Kit, was employed to profile the transcriptome of MeJA induction at thirteen points in time. Real-time PCR was performed to verify changes in expression of 36 unique ESTs (uniESTs), detected via microarray. Genes upregulated by MeJA include those involved in saikosaponin, amino acid, phenylpropanoid and jasmonate biosynthesis, defense signaling, plant hormone regulation and stress protection; indicating that the JA signaling pathway interacts with other signaling pathways leading to transcriptional reprogramming in B. kaoi adventitious roots. Some of these MeJA-induced genes have applications in agricultural, pharmaceutical and food industries.

The male effect is a well-known phenomenon in female sheep and goats whereby a pheromone-induced activation of reproductive function occurs. However, the molecule(s) involved in this phenomenon are unknown. We investigated gene expression profiles for the induction of male effect pheromone synthesis using a PCR-based cDNA subtraction strategy. We constructed two subtracted cDNA libraries using mRNA from the skin of the head or rump region of orchidectomized male goats with or without pheromone induction using testosterone or dihydrotestosterone (DHT). Both libraries were assumed to contain genes whose expression increases with pheromone induction. Clones (n ¼480) from each library were sequenced and identified using BLAST to reveal 115 and 239 types of sequences in the libraries of the head and rump region, respectively. Among these, 12 genes were expressed in both libraries. We conducted real-time PCR to further analyze their expression using cDNA samples derived from pheromone-producing or nonproducing skin from the head of an ovariectomized female goat with or without DHT implantation, respectively. For nine genes, we observed significantly increased expression in samples following DHT implantation. Among these, stearoyl-CoA desaturase 1 (SCD1) and elongation of long chain fatty acids family member 5 (ELOVL5) genes showed more than 100-fold higher expression levels in pheromone-positive samples, suggesting that the products of these genes may be important in pheromone synthesis. gene regulation, implantation, pheromones, seasonalreproduction, testosterone

Following baculovirus infection, the level of host messenger RNA (mRNA) declines and the synthesis of host proteins is shutoff in virus-infected cells. To comprehensively understand the regulation of host gene expression by Bombyx mori nucleopolyhedrovirus (BmNPV), we took a complementary DNA (cDNA) subtraction approach. Northern blot analysis was then performed to confirm whether obtained clones are differentially expressed. From these analyses, we obtained 4 down-, 7 up- and 2 non-regulated host gene candidates during the early stage of infection. Sequence analyses showed that these may encode proteins involved in transcription, cell cycle, cell adhesion, protein degradation, apoptosis or energy metabolism. We then measured the ADP/ATP ratio to address the response of host energy metabolism upon baculovirus infection. The ADP/ATP ratio showed cell death-like rapid increase following BmNPV infection with a transient decrease during 12–24 h post-infection (p.i.). Taken together, our results suggest that general events in host cells are controlled at the level of mRNA expression by BmNPV infection.

Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China.

Hyperplasia of parathyroid glands in patients with chronic renal failure is classified into diffuse (DH) and nodular (NH) types, and NH is often refractory to routine medical therapy.Although it is considered that the parenchymal cells initially proliferate diffusely and then some of them are transformed to form nodules consisting of monoclonal cells, the underlying molecular mechanism for such a transformation is not fully understood. In this study we tried to identify the genes that are up-regulated in NH.The cDNA population prepared from DH was subtracted from that prepared from NH by a PCR-based cDNA subtraction method. The resultant cDNAs were cloned and sequenced. To confirm the up-regulation of the identified genes, a total of 35 parathyroid glands (18 DH, 16 NH, and one mixed) obtained from 21 patients were analyzed.One of the nuclear genes identified was the PRKAR1A gene, which encodes type Ialpha regulatory subunit (RIalpha) of cAMP-dependent protein kinase (PKA). Immunohistochemical analysis demonstrated that RIalpha was abundantly expressed in the nodular region, whereas the adjacent diffuse region displayed relatively low expression. Northern and Western blot analyses demonstrated up-regulation of RIalpha expression in most NH tested. Determination of PKA activities revealed that free PKA activities measured in the absence of cAMP in the assay were inversely correlated with RIalpha expression, indicating the functional significance of RIalpha up-regulation.These results suggest that the aberrant expression of RIalpha is involved in the diffuse to nodular transformation of hyperplasia of parathyroid glands by impairing cAMP/PKA signal transduction.

Matia‰ovic J., P. Kralik, L. Leva, M. Faldyna, P. Hofiin: Suppressive Subtraction Hybridization on Stimulated Primary Horse Macrophages. Acta Vet Brno 75, 2006: 337-341. To study genes potentially involved in genetic resistance to infectious diseases in the horse, suppressive subtraction hybridization was used to identify genes expressed in primary horse macrophages after their stimulation with E. coli. Overnight culture of blood monocyte-derived macrophage cells was stimulated with E. coli K12 in ratio 40 E. coli cells to one macrophage cell. After 4 hours of incubation, non-phagocyted bacteria were washed away. Following next 20 hour incubation in MEM alpha containing 5 µg of gentamycin in 1 ml of media, mRNA was isolated and used in Clontech PCR-Select cDNA Subtraction Kit. Expression of several known horse genes, as well as some new ESTs (expressed sequence tags) showing sequence similarity with immunity-related genes from other species was identified. SSH, horse, monocyte, immunity

The analysis of cell type-specific gene expression is an essential step in understanding certain biological processes during plant development, such as differentiation. Although methods for isolating specific cell types have been established, the application of cDNA subtraction to small populations of isolated cell types for direct identification of specific or differentially expressed transcripts has not yet been reported. As a first step in the identification of genes expressed differentially between maize egg cells and central cells, we have manually isolated these types of cell, and applied a suppression-subtractive hybridization (SSH) strategy. After microarray screening of 1030 cDNAs obtained from the subtracted libraries, we identified 340 differentially expressed clones. Of these, 142 were sequenced, which resulted in the identification of 62 individual cDNAs. The expression patterns of 20 cDNAs were validated by quantitative RT-PCR, through which we identified five transcripts with cell type-specific expression. The specific localization of some of these transcripts was also confirmed by in situ hybridization on embryo sac sections. Taken together, our data demonstrate the effectiveness of our approach in identifying differentially expressed and cell type-specific transcripts of relatively low abundance. This was also confirmed by the identification of previously reported egg cell- and central cell-specific genes in our screen. Importantly, from our analysis we identified a significant number of novel sequences not present in other embryo sac or, indeed, in other plant expressed sequence tag (EST) databases. Thus, in combination with standard EST sequencing and microarray hybridization strategies, our approach of differentially screening subtracted cDNAs will add substantially to the expression information in spatially highly resolved transcriptome analyses.

The main objective of the present study was to identify novel oocyte-specific genes in three different species: bovine, mouse, and Xenopus laevis. To achieve this goal, two powerful technologies were combined: a polymerase chain reaction (PCR)-based cDNA subtraction, and cDNA microarrays. Three subtractive libraries consisting of 3456 clones were established and enriched for oocyte-specific transcripts. Sequencing analysis of the positive insert-containing clones resulted in the following classification: 53% of the clones corresponded to known cDNAs, 26% were classified as uncharacterized cDNAs, and a final 9% were classified as novel sequences. All these clones were used for cDNA microarray preparation. Results from these microarray analyses revealed that in addition to already known oocyte-specific genes, such as GDF9, BMP15, and ZP, known genes with unknown function in the oocyte were identified, such as a MLF1-interacting protein (MLF1IP), B-cell translocation gene 4 (BTG4), and phosphotyrosine-binding protein (xPTB). Furthermore, 15 novel oocyte-specific genes were validated by reverse transcription-PCR to confirm their preferential expression in the oocyte compared to somatic tissues. The results obtained in the present study confirmed that microarray analysis is a robust technique to identify true positives from the suppressive subtractive hybridization experiment. Furthermore, obtaining oocyte-specific genes from three species simultaneously allowed us to look at important genes that are conserved across species. Further characterization of these novel oocyte-specific genes will lead to a better understanding of the molecular mechanisms related to the unique functions found in the oocyte.

To enrich differentially expressed sequence tags (ESTs) for aluminum (Al) tolerance, cDNA subtraction libraries were generated from Al-stressed roots of two wheat (Triticum aestivum L.) near-isogenic lines (NILs) contrasting in Al-tolerance gene(s) from the Al-tolerant cultivar Atlas 66, using suppression subtractive hybridization (SSH). Expression patterns of the ESTs were investigated with nylon filter arrays containing 614 cDNA clones from the subtraction library. Gene expression profiles from macroarray analysis indicated that 25 ESTs were upregulated in the tolerant NIL in response to Al stress. The result from Northern analysis of selected upregulated ESTs was similar to that from macroarray analysis. These highly expressed ESTs showed high homology with genes involved in signal transduction, oxidative stress alleviation, membrane structure, Mg2+ transportation, and other functions. Under Al stress, the Al-tolerant NIL may possess altered structure or function of the cell wall, plasma membrane, and mitochondrion. The wheat response to Al stress may involve complicated defense-related signaling and metabolic pathways. The present experiment did not detect any induced or activated genes involved in the synthesis of malate and other organic acids in wheat under Al-stress. (Managing editor: Ping HE)

In an attempt to understand more about the molecular mechanism by which pollen development is regulated and coordinated in rice, we employed a cDNA subtraction strategy to identify genes that differentially expressed between the wild-type, AN-N and its thermosensitive genic male-sterile mutant (TGMS), AN S-1 during microsporogenesis. Eleven clones exhibiting differential expression patterns were isolated and identified. We report here in detail the molecular cloning and characterization of one of the clones, OS-DFR2 which encodes a dihydroflavonol 4-reductase-like protein. RNA gel blot analysis revealed that OS-DFR2 expressed strongly in anthers but no expression was detected in roots, leaves, stems and pistils, indicating that OS-DFR2 was a tissue-specific gene. Furthermore, the expression of OS-DFR2 was developmentally regulated during early microsporogenesis. It accumulated at high levels in anthers undergoing meiosis and reached a maximal value during tetrad formation stage in the wild-type, AN-N. In AN S-1, the expression of OS-DFR2 resembled that of AN-N except its mRNA level is approximately one-fourth of that found in AN-N at the tetrad formation stage. In situ analysis indicated that OS-DFR2 transcript was specifically expressed in microsporocyte of AN-N; whereas OS-DFR2 was present both in microsporocyte and tapetal cells in AN S-1. The significance and possible role played by OS-DFR2 during male gametophyte development in rice is discussed.

Most previous efforts to isolate genes that are expressed during fruit development have focused on fruit ripening. As a result, information is lacking on fruit genes that are specifically expressed at early developmental stages. Using a cDNA subtraction technique, we isolated fruit-specific genes that are expressed during the cell expansion phase of tomato (Lycopersicon esculentum Mill) fruit development. One of the isolated cDNAs, LeODD, is transiently expressed 15 days after flowering in a nearly fruit-specific manner during the initial period of cell expansion. Southern blot analysis indicated that LeODD is encoded by a single gene. LeODD is homologous to 2-oxoglutarate-dependent dioxygenase genes, and the key amino acid residues in the binding sites for ferrous iron and 2-oxoglutarate are completely conserved. The amino acid sequence identity between LeODD and other 2-oxoglutarate-dependent dioxygenases is relatively low, suggesting that LeODD is a novel enzyme of this family. Another of the isolated cDNAs, LeGLO2, is also highly expressed at 15 days after flowering. LeGLO2 is thought to be a novel glycolate oxidase isoform that functions in fruit. 2-Oxoglutarate, the cosubstrate of LeODD, could be supplied by a LeGLO2-mediated glycolate pathway in immature fruit. The coordinate expression of LeODD and LeGLO2 may play a role in the biosynthesis of a metabolite, such as a plant hormone or secondary metabolite, that is required during the initial period of the cell expansion phase of fruit development.

Heterosis was defined as the advantage of hybrid performance over its parents in terms of growth and productivity. Previous studies showed that differential gene expression between hybrids and their parents is responsible for the heterosis; however, information on systematic identification and characterization of the differentially expressed genes are limited. In this study, an interspecific hybrid between common wheat (Triticum aestivum. L., 2n = 6x = 42, AABBDD) line 3338 and spelt (Triticum spelta L. 2n = 6x = 42, AABBDD) line 2463 was found to be highly heterotic in both aerial growth and root related traits, and was then used for expression assay. A modified suppression subtractive hybridization (SSH) was used to generate four subtracted cDNA libraries, and 748 nonreduandant cDNAs were obtained, among which 465 had high sequence similarity to the GenBank entries and represent diverse of functional categories, such as metabolism, cell growth and maintenance, signal transduction, photosynthesis, response to stress, transcription regulation and others. The expression patterns of 68.2% SSH-derived cDNAs were confirmed by reverse Northern blot, and semi-quantitative RT-PCR exhibited the similar results (72.2%). And it was concluded that the genes differentially expressed between hybrids and their parents involved in diverse physiological process pathway, which might be responsible for the observed heterosis.

Avian infectious bronchitis virus (IBV) infection is one of the major viral respiratory diseases of chickens. Better understanding of the molecular basis of viral pathogenesis should contribute significantly towards the development of improved prophylactic, therapeutic and diagnostic reagents to control infections. In the present investigation, transcriptional profiles were analyzed by using RNA recovered from the lung tissue of IBV infected 18-day-old chicken embryos at 6, 24, 48 and 72 h post IBV infection. This microarray analysis was completed using avian cDNA arrays comprised of fragments of 1191 unique chicken and turkey gene transcripts. These arrays were generated from normalized cDNA subtraction libraries that were derived from avian pneumovirus (APV) infected chicken embryo fibroblast (CEF) cultures and tissues obtained from APV infected turkeys subtracted with their respective uninfected cultures and tissues. Of the 1191 unique genes represented on the array, the expression of a total of 327 genes (27% of total) were altered by two-fold or more from 6 through 72 h post-infection. A comparative analysis of IBV regulated genes with genes previously reported to change in expression following infection with other avian respiratory viruses revealed both conserved and unique changes. Real-time qRT-PCR was used to confirm the regulated expression of genes related to several functional classes including kinases, interferon induced genes, chemokines and adhesion molecules, vesicular trafficking and fusion protein genes, extracellular matrix protein genes, cell cycle, metabolism, cell physiology and development, translation, RNA binding, lysosomal, protein degradation and ubiquitination related genes. Microarray analysis served as an efficient tool in facilitating a comparative analysis of avian respiratory viral infections and provided insight into host transcriptional changes that were conserved as well as those which were unique to individual pathogens.

In patients with chronic myeloid leukemia (CML) who do not reach a (near) complete cytogenetic response, the disease progresses over several years from an indolent, chronic phase into a rapidly fatal blast crisis. Events that are responsible for this transformation process are largely unknown. To identify changes in gene expression that occurred during the course of the disease, we performed cDNA subtraction on sequentially stored peripheral blood mononuclear cell pellets, collected throughout the course of disease of a single CML patient. In total, 32 differentially expressed sequences were identified, of which 27 corresponded to known genes. On quantitative PCR, eight of these genes, YWHAZ, GAS2, IL8, IL6, PBEF1, CCL4, SAT and MMRN, showed comparable differential expression in additional CML patient samples. This set of genes can be considered as a starting point for further research on causes of disease transformation in CML and may lead to new targets in the treatment of resistant CML.

The hepatitis B virus (HBV)-encoded X antigen (HBxAg) may contribute to the development of hepatocellular carcinoma (HCC) through the upregulated expression of selected cellular genes. To identify these genes, RNAs isolated from HBxAg-positive and -negative HepG2 cells were compared by PCR select cDNA subtraction. One gene overexpressed in HBxAg-positive cells by Northern and Western blotting is the ribosomal protein S15a. The S15a mRNA is 535 base pairs, encoding a protein 130 amino acids long with a molecular weight of 14.3 kDa. S15a expression was upregulated in HBV-infected livers, where it costained with HBxAg. Overexpression of S15a stimulated cell growth, colony formation in soft agar, and tumor formation in SCID mice. Hence, HBxAg upregulated the expression of S15a, the latter of which participates in the development of HCC, perhaps by altering the integrity of translation.

The ALP1 [aci-reductone dioxygenase (ARD)-like protein 1] gene was identified in a comprehensive cDNA subtraction aimed at identifying genes regulated by androgens in the rat ventral prostate. ALP1 is homologous to the ARD/ARD' that were discovered in Klebsiella pneumoniae as enzymes that have the same polypeptide sequence and differ only in their metal content. This family of proteins is evolutionarily conserved from bacteria to humans and is involved in the methionine salvage pathway. Northern and Western blot confirmed the regulation of ALP1 by androgens in the rat ventral prostate. ALP1 mRNA is expressed in a variety of tissues; however, its regulation by androgens was specific to the prostate. ALP1 is expressed by the glandular epithelial cells of the rat prostate, with little or no expression in the stromal cells. ALP1 is down-regulated in the different rat Dunning tumor cell lines compared with the normal or castrated rat prostate. Expression studies showed that ALP1 overexpression is not tolerated by AT6.1 cells. Further studies demonstrated that ALP1 is also down-regulated in the human prostate cancer cell lines LNCaP, PC3, and DU145, and overexpression induces cell death in these cells. Taken together, our observations suggest that ALP1 may have an important role in androgen regulated prostate homeostasis as well as in prostate cancer progression by regulating cell death of prostate cancer cells.

We have analyzed the gene expression profile of monocytes in response to a highly purified cell wall fraction of M y cobacterium bovis BCG, a clinically approved adjuvant known as BCG cell wall skeleton (BCG-CWS). It is composed of mycolic acid, arabinogalactan, and peptidoglycan and confers Toll-like receptor 2 (TLR2)- and TLR4-dependent signaling that induces monocytes to differentiate into antigen-presenting cells (APCs). Here we report differential gene expression analysis with BCG-CWS-stimulated versus nonstimulated monocytes. BCG-CWS exerted massive induction of genes regulated by TLR signaling. Marked gene regulatory characteristics in BCG-CWS-stimulated cells compared to lipopolysaccharide (LPS)-stimulated cells follow. (i) Spliced mRNAs encoding soluble forms of TREM-1 and TREM-2 (recently discovered inflammatory-signal-amplifying receptors) were regulated by BCG-CWS, resulting in their differential expression. (ii) The genes for zinc-iron transporter protein (ZIP)-like family proteins HKE-1 and LIV-1 were induced exclusively by BCG-CWS. (iii) Interleukin-23 (IL-23), rather than IL-12p70, was induced by BCG-CWS, while interferon-inducible genes were induced only by LPS. By Northern and reverse transcription-PCR analyses, we confirmed the differential expression of more than 30 BCG-CWS regulatory genes, and their expression was compared with that of LPS and other known TLR ligands. A battery of genes responded rapidly and for a short time to LPS but for a long time to BCG-CWS. Structural analysis of the identified novel or hypothetical proteins revealed that some are potential candidates as signaling mediators or transcriptional regulators. Hence, BCG-CWS may profoundly modulate APC responses in a way distinct from that of LPS, leading to possible advantages for its adjuvant-active therapeutic potential.

Representational difference analysis, a cDNA subtraction approach, was used to identify genes that are expressed in acute leukemia but not in normal hematological tissues. We isolated a cDNA fragment from a cell line derived from a B cell acute lymphoblastic leukemia bearing two Philadelphia chromosomes. The cDNA derives from an orphan gene that was named BLACE. BLACE is located in region 7q36 and encodes a major 5.3-kb transcript and several alternatively spliced minor transcripts. Significant expression of BLACE was detected by RT-PCR and quantitative RT-PCR in bone marrow samples from B cell acute lymphoblastic leukemia patients. BLACE was not or was scarcely expressed in other types of hematological malignancies, in normal tissues, and in solid tumors.

To identify differentially expressed genes in human HCC in China, we applied a modified SSH method for cDNA subtraction. Such modification has made the method more effective for subtraction. We have obtained 36 and 24 differentially expressed cDNA fragments after modified SSH from 4 paired samples of human HCC and non-HCC tissues, respectively. Reverse Northern blotting analysis was performed to further identify the genes differentially expressed in the HCC and non-HCC tissue samples. There were 25 genes really overexpressed in HCC, and their corresponding encoding molecules may reflect the events of cell accelerated metabolism, proliferation, angiogenesis, anti-apoptosis, tumorigenesis (TLH107, TFH9) and the potential for metastasis. Of the 25 genes overexpressed in HCC, 5 were novel and their full-length cDNAs were cloned. These 5 novel genes are functionally associated with the occurrence and development of HCC according to the Database analysis. In the paired non-HCC tissues, there were 15 genes lowly or not expressed in HCC, and their encoding proteins function as tumor suppressors (TFA3, TFA11), acute-phase reactive proteins, and the blood plasma proteins that are mainly or exclusively synthesized in the liver. The distinct profiles of the differentially expressed genes in HCC and the paired non-HCC tissues have partially reflected the genetic alterations during HCC tumorigenesis. The novel HCC-specific gene TLH6 and the CT antigen encoding gene TLH107 may have diagnostic and therapeutic potentials in HCC and/or other solid cancers.

The fungus Cercospora zeae-maydis causes gray leaf spot of maize and produces cercosporin, a photosensitizing perylenequinone with toxic activity against a broad spectrum of organisms. However, little is known about the biosynthetic pathway or factors that regulate cercosporin production. Analysis of a cDNA subtraction library comprised of genes that are up-regulated during cercosporin synthesis revealed a sequence highly similar to mitogen-activated protein (MAP) kinases in other fungi. Sequencing and conceptual translation of the full-length genomic sequence indicated that the gene, which we designated CZK3, contains a 4,119-bp open reading frame devoid of introns and encodes a 1,373-amino acid sequence that is highly similar to Wis4, a MAP kinase kinase kinase in Schizosaccharomyces pombe. Targeted disruption of CZK3 suppressed expression of genes predicted to participate in cercosporin biosynthesis and abolished cercosporin production. The disrupted mutants grew faster on agar media than the wild type but were deficient in conidiation and elicited only small chlorotic spots on inoculated maize leaves compared with rectangular necrotic lesions incited by the wild type. Complementation of disruptants with the CZK3 open reading frame and flanking sequences restored wild-type levels of conidiation, growth rate, and virulence as well as the ability to produce cercosporin. The results suggest that cercosporin is a virulence factor in C. zeae-maydis during maize pathogenesis, but the pleiotropic effects of CZK3 disruption precluded definitive conclusions.

Rice (Oryza sativa L.) is sensitive to chilling particularly during early seedling development. Given the biochemical complexity of tolerance mechanisms, genetic potential for this trait depends on highly coordinated expression of many genes. We used a simple cDNA subtraction strategy to develop Expressed Sequence Tags (ESTs) that represent an important subset of cold stress-upregulated genes. The 3,084 subtracted cDNA clones represent a total of 1,967 unigenes from 1,354 singletons and 613 contigs. As expected in the developing seedlings, genes involved in basic cellular processes, i.e., metabolism, growth and development, protein synthesis, folding and destination, cellular transport, cell division and DNA replication were widely represented. Genes with stress-related and regulatory functions comprised 23.17% of the total ESTs. These categories included proteins with known function in cellular defenses against abiotic (drought, cold and salinity) and biotic (pathogen) stresses, and proteins involved in developmental and stress response signalling and transcription. Based on the types of genes represented, tolerance mechanisms rely on precise integration of developmental processes with stress-related responses. A large fraction of the ESTs (38.7%) represents unknown proteins. This EST library is a rich source of cold stress-related genes, and supplements for other publicly available libraries for comprehensive analysis of the stress-response transcriptome.

The genetics and expression of a lipid transfer protein (LTP) gene was examined during abscission of mature fruit of 'Valencia' orange. A cDNA encoding an LTP, CsLTP, was isolated from a cDNA subtraction library constructed from mature fruit abscission zones 48 h after application of a mature fruit-specific abscission agent, 5-chloro-3-methyl-4-nitro-pyrazole (CMN-pyrazole). A full-length cDNA clone of 652 nucleotides was isolated using 5' and 3' RACE followed by cDNA library screening and PCR amplification. The cDNA clone encoded a protein of 155 amino acid residues with a molecular mass and isoelectric point of 9.18 kDa and 9.12, respectively. A partial genomic clone of 505 nucleotides containing one intron of 101 base pairs was amplified from leaf genomic DNA. Southern blot hybridization demonstrated that at least two closely related CsLTP genes are present in 'Valencia' orange. Temporal expression patterns in mature fruit abscission zones were examined by northern hybridization. Increased expression of CsLTP mRNA was detected in RNA of mature fruit abscission zones 6, 24, 48, and 72 h after application of a non-specific abscission agent, ethephon. Low expression of CsLTP transcripts was observed after treatment of CMN-pyrazole until 24 h after application. After this time, expression markedly increased. The results suggest that CsLTP has a role in the abscission process, possibly by assisting transport of cutin monomers to the fracture plane of the abscission zone or through its anti-microbial activity by reducing the potential of microbial attack.

The pathogenic fungus Candida albicans commonly causes mucosal surface infections. In immunocompromised patients, C. albicans may penetrate into deeper tissue, enter the bloodstream and disseminate within the host causing life-threatening systemic infections. In order to elucidate how C. albicans responds to the challenge of a blood environment, we analysed the transcription profile of C. albicans cells exposed to human blood using genomic arrays and a cDNA subtraction protocol. By combining data obtained with these two methods, we were able to identify unique sets of different fungal genes specifically expressed at different stages of this model that mimics bloodstream infections. By removing host cells and incubation in plasma, we were also able to identify several genes in which the expression level was significantly influenced by the presence of these cells. Differentially expressed genes included those that are involved in the general stress response, antioxidative response, glyoxylate cycle as well as putative virulence attributes. These data point to possible mechanisms by which C. albicans ensures survival in the hostile environment of the blood and how the fungus may escape the bloodstream as an essential step in its systemic dissemination.