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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.

Sesamum indicum is widely cultivated in India for its superior agronomic characters like adaptation to the warm climate of India and resistance to drought and pathogen attack. However, the yield of sesame per unit area of land is quite low. In this context, the genes involved with reproductive tissue development is very important since they are directly involved with crop productivity. Therefore, identification and isolation of anther-specific genes through PCR-Select cDNA subtraction between cDNAs of anthers and leaves was carried out. Sequencing of ~ 700 clones from the cDNA subtraction library followed by BLASTX analysis using sesame database resulted in the identification of 163 annotated unique transcripts, of which 18 transcripts showed homology with the genes of exclusive anther-/pollen-specific functions. Furthermore, anther-specific expression of some of the sesame transcripts as well as already identified anther-specific genes of Arabidopsis and rice have been monitored during development of sesame anthers. Subsequently, full-length cDNA sequence and the upstream sequence of an anther-specific β-1 3-glucanase gene was isolated. Transgenic tobacco plants were developed with GUS gene under the control of a 437-bp upstream sequence along with the first exon and first intron of the gene. Monitoring of GUS gene expression pattern indicated exclusive expression of the promoter in the tapetal cell layer before tetrad formation in the anther. Interestingly, upon treatment with ABA as well as drought stress, GUS was found to express in the vegetative parts of the plant such as leaves and roots as well. [ABSTRACT FROM AUTHOR]

For many contaminants, biomarker genes are unknown or assays are unavailable, and most biomarker assays target the first pathway step. Herein, we obtained sequences for all of the genes in a previously hypothesized o -xylene degradation pathway based on similarities to analogous genes in a known toluene degradation pathway. Comparative metatranscriptomics resulted in sequences for genes annotated as bssA, bbsEF, bbsCD, and bbsB, while genes for bbsG and bbsH were notably missing. Prokaryotic Suppressive Subtractive Hybridization PCR cDNA Subtraction (Prokaryotic SSH-PCR cDNA Subtraction) was applied for the first time to a mixed-species microbiome to enrich abundances of genes up-regulated during o -xylene degradation prior to metatranscriptomic sequencing. The subtracted metatranscriptome was sequenced using the MinION; this approach was highly effective at retrieving sequences for biodegradation genes including the previously missing bbsG and bbsH. Reverse transcription quantitative PCR (RT-qPCR) analysis confirmed up-regulation. Thus, data reported herein lend credence to the previously hypothesized anaerobic o -xylene degradation pathway, and new biomarker assays are presented. A novel biomarker development tool for mixed species systems, Subtractive Community Metatranscriptomics (SCM), is demonstrated. [Display omitted] • cDNA Subtraction was applied for the first time to a mixed microbiome. • Biodegradation pathway genes for anaerobic o -xylene are reported. • RT-qPCR data show all pathway genes except bbsG and bbsH are highly upregulated. • cDNA Subtraction has advantages over metatranscriptomics for biomarker retrieval. [ABSTRACT FROM AUTHOR]

Abstract: Astrocytes associated with beta-amyloid (Aβ)-deposited senile plaques are a common neuropathological feature of Alzheimer''s disease (AD). There is little doubt that the association of Aβ with the major component in the central nervous system cells significantly influences disease progression, however, the molecular mechanisms by which Aβ contributes to the astrocyte-mediated neuropathological changes have not been well established. In an effort to identify astrocyte-derived molecules that may be closely associated with exacerbation of AD, we identified a novel Aβ-induced rat gene, whose mouse counterpart, mitsugumin 29 (MG29), is known to be involved in intracellular Ca2+ homeostasis in skeletal muscle. To evaluate the roles of human MG29 in AD, we investigated its expression in AD brain. In non-AD brains, MG29 mRNA has been detected in neurons, but not in quiescent astrocytes. On the contrary, in AD brains, MG29 is expressed in activated astrocytes associated with senile plaques, but not expressed in neurons around lesions. Human MG29-transfected cells express the protein in the endoplasmic reticulum and the level of expression is involved in Ca2+-dependent exocytosis mechanisms. Increased MG29 expression in senile plaques-associated activated astrocytes suggests that intracellular Ca2+ turnovers may be changed in these astrocytes. This might be related in sustained Ca2+-dependent exocytosis of various transmitters including glutamate, leading to the acceleration of neurodegeneration in the lesion observed in AD. [Copyright &y& Elsevier]

Abstract: This is the first report describing the expression of canine calreticulin (cCRT) in canine mammary gland tumour (MGT). Using cDNA subtraction method, it is found that mRNAs of CRT, cathepsin A, ovostatin, and lactotransferrin were differentially expressed in mammary adenocarcinoma as compared to hyperplasia, both of which were obtained from the dog. Furthermore, the mRNA expression levels of CRT and cathepsin A were significantly higher in canine MGT samples than in nontumour samples. In contrast, immunohistochemical studies have indicated that the expression of cCRT protein found to be detected in most of mammary gland tissues and was not correlated to the types of canine MGTs. Furthermore, cCRT was molecularly cloned, and the amino acid sequence of cCRT was found to be very similar to those of other species. Further studies are required to elucidate additional roles of cCRT in canine MGT. [Copyright &y& Elsevier]

Primary objective: The immunosuppressant cyclosporin A (CsA) is reported to have a strong anti-ischemic effect. Although this neuroprotective effect is speculated to be related to the blockade of a mitochondrial permeability transition pore (mPTP), the underlying molecular mechanism remains to be elucidated. This study focused on the effect of CsA on transcriptional regulation in brain cells. Methods: CsA and a control substance were injected into rat brains and purified extracted mRNA. Both mRNAs were compared using a cDNA subtraction technique. Results: Nine significantly up-regulated genes and seven significantly down-regulated genes were detected following CsA administration. All of the up-regulated genes are neurotrophic or reported to have roles in regeneration of brain tissue. Among the down-regulated genes, three are known to be detrimental to neuronal cells and are also reported to facilitate the pathology of Alzheimer's disease (AD) and four genes are related to oxidative metabolism. Conclusions: Strong immunosuppression would present as a side-effect during CsA use as a neuroprotectant. The results of this study will help to discriminate between the CsA immunosuppressive effect and the neuroprotective effect at the molecular level and may lead to the development of new conceptual and pharmacological tools. [ABSTRACT FROM AUTHOR]

Ovarian cancer, one of the most common gynecological malignancies, has an aggressive phenotype. It is necessary to develop novel and more effective treatment strategies against advanced disease. Protein tyrosine kinases (PTKs) play an important role in the signal transduction pathways involved in tumorigenesis, and represent potential targets for anticancer therapies. In this study, we performed cDNA subtraction following polymerase chain reaction (PCR) using degenerate oligonucleotide primers to identify specifically overexpressed PTKs in ovarian cancer. Three PTKs, janus kinase 1, insulin-like growth factor 1 receptor, and discoidin domain receptor 1 (DDR1), were identified and only DDR1 was overexpressed in all ovarian cancer tissues examined for the validation by quantitative real-time PCR. The DDR1 protein was expressed in 63% (42/67) of serous ovarian cancer tissue, whereas it was undetectable in normal ovarian surface epithelium. DDR1 was expressed significantly more frequently in high-grade (79%) and advanced stage (77%) tumors compared to low-grade (50%) and early stage (43%) tumors. The expression of the DDR1 protein significantly correlated with poor disease-free survival. Although its functional role and clinical utility remain to be examined in future studies, our results suggest that the expression of DDR1 may serve as both a potential biomarker and a molecular target for advanced ovarian cancer. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Bovine growth hormone (bGH) transgenic mice develop severe kidney damage. This damage may be due, at least in part, to changes in gene expression. Identification of genes with altered expression in the bGH kidney may identify mechanisms leading to damage in this system that may also be relevant to other models of kidney damage. Design: cDNA subtraction libraries, northern blot analyses, microarray analyses and real-time reverse transcription polymerase chain reaction (RT/PCR) assays were used to identify and verify specific genes exhibiting differential RNA expression between kidneys of bGH mice and their non-transgenic (NT) littermates. Results: Immunoglobulins were the vast majority of genes identified by the cDNA subtractions and the microarray analyses as being up-regulated in bGH. Several glycoprotein genes and inflammation-related genes also showed increased RNA expression in the bGH kidney. In contrast, only a few genes were identified as being significantly down-regulated in the bGH kidney. The most notable decrease in RNA expression was for the gene encoding kidney androgen-regulated protein. Conclusions: A number of genes were identified as being differentially expressed in the bGH kidney. Inclusion of two groups, immunoglobulins and inflammation-related genes, suggests a role of the immune system in bGH kidney damage. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented regarding the study on correlation of palmoplantar collagen bundle size with a characteristic spatial expression of proteoglycans decorin and lumican.

Abstract: The information conveyed from dendritic cells (DCs) to naïve CD4+ T cells has crucial influence on their differentiation toward effector T cells. In an effort to identify DC-derived molecules directly contributing to T cell differentiation, we searched for molecules distinctively expressed between two DC subtypes, which were differentiated from peripheral monocytes by cultivation with GM-CSF (for DC1) or IL-3 (for DC2) in the presence of IL-4 and had the ability to induce naïve T cells to differentiate into Th1 or Th2 cells, respectively. As the first step to address this issue, we subtracted DC1 transcripts from those of DC2 and compiled the gene profile dominantly expressed in DC2, whose products are known to reside in other than the nucleus. Intriguingly, many of them were molecules involved in Th2-skewed disease pathologies, such as FN1, ITGAE, GPNMB, PLAUR, FPRL2, LILRB4, SERPINE1, ALOX15, TBXAS1, NCF2, CCL3, IL1RN, SPARC, and STAB1, suggesting that DCs function not only as antigen presenting cells but also as producers of Th2 pathology specific milieus leading to disease deteriorations. We also found that expressions of CYP27A1, PPAP2B, RSAD2, and ABCC3 were up-regulated in DC2, implying their significant function in Th2-deviated states. The identification of differentially expressed genes between DC subtypes provides new insights into their functions and our comparative gene expression profile will be highly useful for the identification of DC-derived key molecules for T cell differentiation. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: The rat prostate is dependent on androgen for growth and differentiation. In an effort to find novel genes involved in androgen-induced growth of the rat prostate, we carried out PCR-based subtractive hybridization and identified several factors that were transiently up-regulated after androgen stimulation in castrated rat prostate. Among them, a novel member of the Spot 14 family has been identified. This protein (Spot 14-like androgen-inducible protein, SLAP) exhibited the highest homology (about 50%) with zebrafish gastrulation-specific G12 protein and about 30% homology with rat Spot 14. The SLAP mRNA level decreased following castration and transiently increased after testosterone replacement, attaining a peak 3 days after the treatment. The change in the protein level was similar to that in mRNA except that it was low in both untreated and castrated rat prostate tissue. In normal adult rats, SLAP was expressed at relatively high levels in the lung, stomach and liver. Unlike the prostate, SLAP expression in the lung was not affected by the androgen status. Like other members of the Spot 14 family, SLAP has a leucine-zipper motif in its C-terminal region, making it possible to form a stable homodimer. Though the physiological role of SLAP remains to be clarified, the current results suggest that SLAP expression is associated with some growth-related processes in regrowing rat prostate. [Copyright &y& Elsevier]

Abstract: The greater horseshoe bat (Rhinolophus ferrumequinum) is a widely distributed small mammal that hibernates annually. A systematic study was initiated to identify differentially expressed genes in hibernating and aroused states of the greater horseshoe bat brain by using suppressed subtractive hybridization technique and dot blot. Forty-one over-expressed ESTs in the hibernating state were found and 17 were known genes reported in NCBI. Among these 17 genes, three were further checked by real time PCR. The bioinformatics analysis suggests that the major over-expressed ESTs may be responsible for the regulation of cell cycle and apoptosis, the growth of neurons, signal transduction and neuroprotection, gene expression regulation, and intracellular trafficking. [Copyright &y& Elsevier]

Abstract: Entomopathogenic nematode Heterorhabditis bacteriophora infective juveniles (IJs) employ multiple strategies to combat with insect innate immune system after invasion. We employed suppressive subtractive hybridization (SSH) technique to study the gene expression in the IJs upon contact with the insect hemolymph in vitro. We identified two clones having higher expression levels in the IJs than IJs treated with insect hemolymph. The differential expression levels were confirmed by Northern blot hybridization with reference to the constitutive expression level of Heterorhabditis bacteriophora actin2 gene. Clone HbGPS11.C1G02 encoded a phosphofructokinase (PFK) with a 2.5kb transcript and clone HbGPS11.C4C08 corresponded to a 2.1kb transcript encoding a protein that had weak similarity to trans-sialidase from Trypanosoma cruzi. The differential expression of PFK in H. bacteriophora IJs before and during recovery process represented a switch from active movement in search of insect hosts to a state of combating insect innate immune system. This first report of H. bacteriophora differential gene expression provides a glimpse at the gene expression profile of H. bacteriophora IJ recovery process. [Copyright &y& Elsevier]

Abstract: Growth hormone (GH) transgenic amago salmon (Oncorhynchus masou) were generated with a construct containing the sockeye salmon GH1 gene fused to the metallothionein-B (MT-B) promoter from the same species. This transgene directed significant growth enhancement with transgenic fish reaching approximately four to five times greater weight than control salmon in F2 and F3 generations. This drastic growth enhancement by GH transgene is well known in fish species compared with mammals, however, such fish can show morphological abnormalities and physiological disorders like other GH transgenic animals. GH is known to have many acute effects, but currently there are no data describing the chronic effects of over-expression of GH on various hepatic genes in GH transgenic fish. Hepatic gene expression is anticipated to play very important roles in many physiological functions and growth performance of transgenic and control salmon. To examine these effects, we performed subtractive hybridization (using cDNA generated from liver RNA) in both directions to identify genes both increased and decreased in transgenic salmon relative to controls (576 clones were isolated and sequenced in total). Heme oxygenase, vitelline envelope protein, Acyl-coA binding protein, NADH dehydrogenase, mannose binding lectin-associated serine protease, hemopexin-like protein, leucyte-derived chemotaxin2 (LECT2), and many other genes were obtained in higher clone frequencies suggesting enhanced expression. In contrast, complement C3-1, lectin, rabin, alcohol dehydrogenase, Tc1-like transposase, Δ6-desaturase, and pentraxin genes were obtained in lower frequencies. Microarray analysis was also performed to obtain quantitative expression data for these subtracted cDNA clones. Analysis of fish across seasons was also conducted using both F2 and F3 salmon. Results of the microarray data essentially corresponded with those of the subtraction data when both F2 and F3 fish were completely immature, but the expression pattern was changed when fish approached maturation. Genes showing enhanced expression in GH transgenic fish in F2 and F3 by array analysis were vitelline envelope protein, hemopexin-like protein, heme-oxygenase, inter α-trypsin inhibitor, LECT2, GTP cyclohydrolase I feedback regulatory protein (GFRP), and bikunin. Reduced expression genes were lectin, Δ6-desaturase, apolipoprotein, and pentraxin. In particular, lectin was found to be highly suppressed in all F2 and immature F3 salmon. Further, serum lysozyme activity, one of innate immunity, was significantly (p <0.05) decreased in both F2 and F3 GH transgenic fish. These results indicate that the GH transgene fish had altered hepatic gene expression relating to iron-metabolism, innate immunity, reproduction, and growth. [Copyright &y& Elsevier]

Abstract: Alfalfa leaf protoplast-derived cells can develop into somatic embryos depending on the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) in the initial culture medium. In order to reveal gene expression changes during the establishment of embryogenic competence, we compared the cell types developed in the presence of 1 and 10 μM 2,4-D, respectively, at the time of their first cell divisions (fourth day of culture) using a PCR-based cDNA subtraction approach. Although the subtraction efficiency was relatively low, applying an additional differential screening step allowed the identification of 38 10 μM 2,4-D up-regulated transcripts. The corresponding genes/proteins were annotated and representatives of various functional groups were selected for more detailed gene expression analysis. Real-time quantitative PCR (RT-QPCR) analysis was used to determine relative expression of the selected genes in 2,4-D-treated leaves as well as during the whole process of somatic embryogenesis. Gene expression patterns confirmed 2,4-D inducibility for all but one of the 11 investigated genes as well as for the positive control leafy cotyledon1 (MsLEC1) gene. The characterized genes exhibited differential expression patterns during the early induction phase and the late embryo differentiation phase of somatic embryogenesis. Genes coding for a GST-transferase, a PR10 pathogenesis-related protein, a cell division-related ribosomal (S3a) protein, an ARF-type small GTPase and the nucleosome assembly factor family SET protein exhibited higher relative expression not only during the induction of somatic embryogenesis but at the time of somatic embryo differentiation as well. This may indicate that the expression of these genes is associated with developmental transitions (differentiation as well as de-differentiation) during the process of somatic embryogenesis. [Copyright &y& Elsevier]

Abstract: Beta-amyloid (Aβ) deposition and senile plaque-associated astrocytes are common neuropathological features of Alzheimer''s disease (AD). Although the molecular mechanisms by which Aβ contributes to the progression of neuropathologic changes have not been entirely established, there is little doubt that the association of Aβ with astrocytes, the predominant cell type in brain, significantly influences exacerbation of the disease. In an effort to identify astrocyte-derived molecules that may be intimately associated with progression of AD, we identified a novel Aβ-induced rat gene, designated Mib, whose human counterpart covers KIAA0233. Mib-transfected C6 cells express Mib protein in the endoplasmic reticulum and endplasmic reticulum–Golgi-intermediate compartment. To evaluate roles of Mib in AD, we investigated its expression in the AD brain. In non-AD brains, Mib mRNA has been detected in neurons but not in quiescent astrocytes. On the contrary, in AD brains, Mib mRNA is expressed in activated astrocytes associated with senile plaques, but not expressed in neurons around lesions. From these observations, Mib appears to be a novel Aβ-responsive gene that may play a role in astrocyte inflammatory activation around senile plaques in the AD brain. [Copyright &y& Elsevier]

Abstract: The microcell-mediated transfer of a normal human chromosome 3 induces replicative senescence in otherwise immortal renal cell carcinoma cells. To identify the genes involved in the chromosome 3-induced cellular mortality, we previously performed a cDNA subtraction experiment using the immortal renal cell carcinoma cells (RCC23) and the mortal counterpart with the transferred chromosome 3 (RCC23+3). We here report the cDNA cloning and characterization of one of the differentially expressed genes, which encodes KIAA1199 protein of unknown function. Northern blot and RT-PCR analyses revealed striking upregulation of KIAA1199 mRNA in mortal RCC23+3 compared with immortal RCC23. However, no significant change in KIAA1199 mRNA expression was observed during replicative aging in vitro (from early passage culture to senescent culture) of mortal human cells including RCC23+3, normal fibroblasts and prostate epithelial cells. Interestingly, an immortal fibroblast cell line and two breast cancer cell lines expressed much lower amounts of KIAA1199 mRNA than their normal counterparts. KIAA1199 mRNA is expressed in a wide range of normal human tissues, with the highest level of expression in brain. The gene is located on chromosome band 15q25, where a brain tumor suppressor gene has been mapped. These findings suggest that KIAA1199 gene may play a role in cellular mortality of normal human cells, which counters cell immortalization and carcinogenesis. [Copyright &y& Elsevier]

The phloem-feeding by greenbug ( Schizaphis graminum) elicits unique interactions with their host plants. To investigate the expression profiles of sorghum genes responsive to greenbug feeding, two subtractive cDNA libraries were constructed through different combinatorial subtractions in a strong greenbug resistance sorghum M627 line and a susceptible Tx7000 line with or without greenbug infestation. A total of 3,508 cDNAs were selected from the two cDNA libraries, and subsequent cDNA microarray and northern blot analyses were performed for identification of sorghum genes responsive to greenbugs. In total, 157 sorghum transcripts were identified to be differentially expressed by greenbug feeding. The greenbug responsive genes were isolated and classified into nine categories according to the functional roles in plant metabolic pathways, such as defense, signal transduction, cell wall fortification, oxidative burst/stress, photosynthesis, development, cell maintenance, abiotic stress, and unknown function. Overall, the profiles of sorghum genes, responsive to greenbug phloem-feeding shared common identities with other expression profiles known to be elicited by diverse stresses, including pathogenesis, abiotic stress, and wounding. In addition to well-known defense related regulators such as salicylic acid, jasmonic acid, and abscisic acid, auxin and gibberellic acid were also involved in mediation of the defense responses against greenbug phloem-feeding in sorghum. [ABSTRACT FROM AUTHOR]

Abstract: Anuran tadpoles (Rana pirica) are induced to develop a higher tail and a bulgy body as predator-specific morphological responses when they are exposed to predatory larval salamanders. Subtractive hybridization was performed using induced tadpole body skin and normal tadpoles’ body skin. A total of 196 clones showed higher expression, and 104 clones showed lower expression, when they formed bulgy bodies. In the subtraction, carboxypeptidase B, trypsinogen, elastase I, fibrinogen, elastase II, triacyl-glycerol lipase, and α1-antitrypsin genes showed lower expression. In contrast, RT-like protein, bullous pemphigoid antigen, phosphoserine aminotransferase, uromodulin, tetranectin, chaperonin-like protein, zinc finger protein, osteonectin, aldehyde dehydrogenase, Sec 23A protein, and ribosomal protein showed higher gene expression. Microarray analysis was also performed using this subtracted cDNA (nine replicates). Results of the microarray data essentially corresponded with those of the subtraction data, and the degree of the suppressed genes was much stronger than that of the expressed genes. Carboxypeptidase B showed the strongest suppression, and its inhibition range was from 1/100 to 3/100 compared with that of control body skin. Strong suppression was also observed with trypsinogen, elastase I, fibrinogen, and elastase II as well. These results can be interpreted as increases of fibrinolysis by strong depression of both carboxypeptidase B and other genes simultaneously, resulting in the retention of blood vessels and facilitating the circulation of blood. Expression was observed in phosphoserine aminotransferase, aldehyde dehydrogenase, RT-related protein, chaperonin-like protein, tetranectin, bullous pemphigoid antigen, uromodulin, and Sec 23A protein. They were significantly (p<0.05) increased and were at least 1.5 times greater compared with the control. From the appearance, it seems that the bulgy shaped body is highly connecting to the bullous pemphigoid (BP) antigen that causes the skin blistering disorder, and tetranectin and uromodulin may be related to the extracell matrix through myogenesis, protein secretion, and ion transport, respectively. Since the RT-related protein gene derived from retrotransposon (L1) is known to disrupt mammalian transcriptomes, retrotransposon may be involved with phenotypic plasticity for morphological defense by Rana prica against predator threat. [Copyright &y& Elsevier]

Abstract: Representational difference analysis of cDNAs (cDNA-RDA) is a sensitive subtractive hybridization technique capable of isolating rare mRNAs differentially expressed in two cell populations. cDNA-RDA can detect sequences represented at 0.0001% in the starting mRNA. By using reverse transcriptase polymerase chain reaction (PCR), cDNA-RDA also lends itself to studies in which samples are derived from limited numbers of cells. Standard cDNA-RDA protocols depend upon the presence of specific restriction enzyme sites in each cDNA, typically enzymes with four base recognition sequences. These sites are used to reduce the cDNA size range and provide primer sites for subsequent PCR amplification. Consequently, transcripts containing fewer than two of the chosen restriction sites are undetectable by cDNA-RDA. We have developed a restriction enzyme site-independent cDNA-RDA protocol called modified RDA (MRDA). We constructed MRDA test sequences from random hexamer-primed cDNA, thereby increasing the representation of mRNAs which are excluded by cDNA-RDA protocols. MRDA is also more efficient than cDNA-RDA at removing highly expressed housekeeping genes during the subtractive hybridization process, thereby allowing more efficient isolation of preferentially expressed mRNAs. Using MRDA, we isolated cDNAs differentially expressed between limited numbers of human CD4+ naive and memory T lymphocyte subsets and skin- and gut-homing memory T cell subsets. [Copyright &y& Elsevier]

The otic vesicle (otocyst) occupies a pivotal position in inner ear development, bridging the gap between otic placode determination, and morphogenesis of vestibular and auditory compartments. The molecular mechanisms underlying the progressive subdivision of the developing inner ear into different compartments, and the molecular control and execution of the different developmental processes involved, are largely unknown. Since relatively few genes have been implicated in these processes, we have undertaken this study to identify genes involved in these early embryonic stages. We have used cDNA subtractions of mouse otic vesicle against adult liver cDNA, and describe a set of 280 candidate genes. We have also performed otic vesicle RNA hybridizations against DNA chips to not only confirm the efficacy of the library approach, but also to investigate the utility of DNA array alternatives. To begin to dissect potential developmental roles, we investigated the spatial pattern of gene expression for a selected set of 80 genes in developing mouse embryos at mid-gestation by whole-mount in situ hybridization. These data illustrate the compartmentalisation of gene expression in the otic vesicle for the majority of genes tested, and furthermore, implicate many of the genes tested with distinct developmental subprocesses. [Copyright &y& Elsevier]

Although the gross embryology of inner ear development has been documented for several different vertebrate species at a descriptive level, our understanding of the molecular mechanisms involved remains rudimentary. Therefore, we have used cDNA subtraction and normalization procedures to define genes upregulated in the 13.5dpc mouse inner ear, a developmental stage where inner ear morphogenesis and tissue remodeling is active and differentiation of future hair cells is being initiated. We recovered 33 different genes from this subtraction and using gene-specific primers have confirmed the transcriptional upregulation of 26 of these in the 13.5dpc inner ear. Northern analyses were used to investigate splicing differences between the inner ear and the whole embryo at 13.5dpc. Spatial localization of expression was determined through whole-ear in situ hybridization analysis, and selected genes were analyzed in more detail through in situ hybridization of tissue sections. These data illustrate that the genes isolated in this study are expressed in the developing otic capsule and/or neuroepithelium. Furthermore, the expression patterns also reveal molecular heterogeneity in the developing capsule and indicate that for some genes, the chondrogenic otic capsule is composed of distinct domains of gene expression. [Copyright &y& Elsevier]

The handedness of gastropods is genetically determined. The freshwater gastropod Lymnaea stagnalis is a normally dextral species, but contains minor sinistral populations. The gene responsible for handedness determination in this species is predicted to function maternally and specifically in the dextral-ovipositing snail. In this study, we used differential screening and cDNA subtraction to isolate eight “dextral genes” that are specific to, or enriched in, the dextral-ovipositing strains of L. stagnalis. These genes were promising candidates for the handedness-determining gene. In order to determine whether the true handedness-determining gene was among them, we tested for genetic correlations between the level of expression of each dextral gene and the handedness phenotype, i.e., the chirality of the next generation offspring, by using a collection of backcross F2 progeny of F1 offspring from crosses between dextral and sinistral strains. Although the present study could not identify the handedness-determining molecules, this approach appears to be promising for the isolation of such developmentally important genes. [ABSTRACT FROM AUTHOR]

Signalling through the fibroblast growth factor family (FGF) of ligands is essential for normal mammalian embryonic development. At a cellular level, many details of the molecular basis of the signal transduction process have been uncovered, but our knowledge of the identity of the downstream effectors of the FGF signal in the developing embryo remains limited. We have used two independent approaches to begin to identify downstream targets of FGF signalling in the embryo: (1) a gene trap approach and (2) cDNA subtraction, using mouse embryonic stem (ES) cells as a cellular system representative of an early window on the developing embryo. Both approaches led to the identification of a number of targets of FGF signalling, and we provide data to show that the chaperone Mrj, the tumour antigen Tum, collapsin mediator response protein Crmp, a novel transcriptional repressor Nac1 and ribophorin are all differentially regulated following FGF signalling. Independent gene trapping of Mrj previously indicated a role for the gene in embryogenesis [Development 126 (1999) 1247], and we present transcript data implicating a number of the newly isolated FGF target genes in different embryonic processes. [Copyright &y& Elsevier]

Mossman virus (MoV) was isolated on two occasions from wild rats trapped in Queensland, Australia, during the early 1970s. Together with Nariva virus and J-virus MoV belongs to a group of novel paramyxoviruses isolated from rodents during the last 40 years, none of which had been characterized at the molecular level until now. cDNA subtraction strategies used to isolate virus-specific cDNA derived from both MoV-infected cells and crude MoV pellets were pivotal steps in rapid characterization of the complete genome sequence. Analysis of the full-length genome and its encoded proteins confirmed that MoV is a novel member of the subfamily Paramyxovirinae which cannot be assigned to an existing genus. MoV appears to be more closely related to another unclassified paramyxovirus Tupaia paramyxovirus (TPMV), isolated from the tree shrew Tupaia belangeri. Together with Salem virus (SalV), a further unclassified paramyxovirus that was isolated from a horse, MoV and TPMV make up a new collection of paramyxoviruses situated evolutionally between the genus Morbillivirus and the newly established genus Henipavirus. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

To identify novel genes that are expressed specifically or preferentially in the cochlea, we constructed a cDNA library enriched for human cochlear cDNAs using a suppression subtractive hybridization technique. We analyzed 2640 clones by sequencing and BLAST similarity searches. One hundred and fifty-five different cDNA fragments mapped in nonsyndromic hearing impairment loci for which the causative gene has not been cloned yet. Approximately 30% of the clones show no similarity to any known human gene or expressed sequence tag (EST). Clones mapping in nonsyndromic deafness loci and a selection of clones that represent novel ESTs were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) of RNA derived from 12 human fetal tissues. Our data suggest that a quarter of the novel genes in our library are preferentially expressed in fetal cochlea. These may play a physiologically important role in the hearing process and represent candidate genes for hereditary hearing impairment. [Copyright &y& Elsevier]

In solid-state cultures (SC), Aspergillus oryzae shows characteristics such as high-level production and secretion of enzymes and hyphal differentiation with asexual development which are absent in liquid (submerged) culture (LC). It was predicted that many of the genes involved in the characteristics of A. oryzae in SC are differentially expressed between SC and LC. We generated two subtracted cDNA libraries with bi-directional cDNA subtractive hybridizations to isolate and identify such genes. Among them, we identified genes upregulated in or specific to SC, such as the AOS (A. oryzae SC-specific gene) series, and those downregulated or not expressed in SC, such as the AOL (A. oryzae LC-specific) series. Sequencing analyses revealed that the AOS series and the AOL series contain genes encoding extra- and intracellular enzymes and transport proteins. However, half were functionally unclassified by nucleotide sequences. Also, by expression profile, the AOS series comprised two groups. These gene products' molecular functions and physiological roles in SC await further investigation. [ABSTRACT FROM AUTHOR]

Calcitonin (CT) is a polypeptide hormone and has a variety of functions including regulation of urinary calcium excretion. By using a cDNA subtraction hybridization method, we identified that NF-IL3A and urokinase-type plasminogen activator (uPA) genes were up-regulated by CT in porcine renal cell line LLC-PK1. CT-mediated induction of these genes was not inhibited by cycloheximide. These data suggest that these up-regulations are not induced by increased synthesis of regulating proteins; therefore, they are immediately response early (IE). We also found that CT treatment led to the phosphorylation of Erk1/2. We demonstrated that PD98059, a MEK1 inhibitor, inhibited CT-induced mRNA expressions of uPA, but had no obvious influence on the NF-IL3A induction. These results demonstrated the inductions of uPA by CT involve Erk1/2 phosphorylation. We provide the first evidence that NF-IL3A expression is up-regulated by CT. The present findings suggest that the transcriptions of the NF-IL3A and uPA could be induced by CT and might be important mediators of CT function in renal cells. [Copyright &y& Elsevier]

β-Amyloid (Aβ) deposition and senile plaque-associated astrocytes are common neuropathological features of Alzheimer''s disease (AD). Although the molecular mechanisms by which Aβ contributes to the progression of neuropathologic changes have not been established entirely, there is little doubt that the association of Aβ with astrocytes, the predominant cell type in brain, has significant influence on exacerbation of the disease. In an effort to identify key molecules involved in AD, we investigated Aβ-responsive genes using rat astrocytes. In this study, we identified a novel Aβ-induced rat gene, designated as Lib, encoding a type I transmembrane protein with an extracellular domain that contains fifteen leucine-rich repeats (LRRs). Human counterpart of rat Lib is located on chromosome 3q29 and human Lib mRNA found in particularly placenta. Lib mRNA levels in rat C6 astrocytoma cells can be increased by pro-inflammatory cytokines and the rat Lib-transfected cells express Lib protein on the cell surfaces. Lib appears to be a member of the LRR superfamily which is involved in cell–cell and/or –extracellular matrix interactions including adhesion or target recognition in neuroinflammatory states. [Copyright &y& Elsevier]

The mechanisms of sexual differentiation of the brain by sex steroids seem to be conserved throughout the mammalian species, although there may be some species differences. In rats, sex-dependent differentiation of the brain occurs in a sex steroid-dependent manner during the perinatal period known as the critical period. Androgen exposure during the perinatal period results in the development of structural and functional sexually dimorphic characteristics in the brain; the absence of testicular androgen leads the central nervous system to develop passively in a primarily female fashion, while the presence of androgen induces the masculinization of the brain. We attempted to characterize sex steroid-inducible genes that are involved in the sexually dimorphic function of the brain. Following the cDNA subtraction between hypothalami of 5-day-old intact and neonatally androgenized female rats, a granulin (grn) precursor gene was identified. The grn gene encodes a 6-kDa polypeptide known as a growth modulating factor of epithelial cells in vitro. Exogenous estrogen, as well as androgen, induced grn gene expression in the neonatal hypothalamus. In the brain of a 5-day-old male rat, grn mRNA was expressed in the ventromedial hypothalamic nucleus and the arcuate nucleus of the hypothalamus. Throughout the critical period for sexual differentiation of the brain, grn gene expression remained high in males, while in females it gradually decreased. Antisense oligodeoxynucleotide (ODN) complementary to grn mRNA was synthesized and infused into the third ventricle of male rats at 2 days of age. Two different control treatments were used; the first consisted of a control sequence ODN that had virtually no homology to known mRNAs, and the second consisted of vehicle alone. After maturation, the subject animals that were treated with antisense ODN of grn displayed significantly lower scores than the control males in various parameters assessing sexual behavior, i.e., mount, intromission, and ejaculation. The present results suggest that the grn gene, the expression of which is induced by sex steroids in the neonatal hypothalamus, plays a crucial role in the functional masculinization of the rat brain. [Copyright &y& Elsevier]

Rat hepatoma Fao is a well-differentiated cell line that expresses numerous liver-specific enzymes and liver-enriched transcription factors, whereas the C2 cell line derived from Fao exhibits dedifferentiated phenotypes and fails to express these differentiated traits. Hence, to identify the gene related to the differentiation status in these cells, we analyzed differentially expressed genes between Fao and C2 cells using a microarray of 1,000 identified cDNA clones, and isolated a cDNA clone from subtractive library constructed by suppression subtractive hybridization. D6.1A, a member of the tetraspanin protein superfamily, was identified as a gene enhanced in C2 cells compared with Fao cells. Interestingly, expression of this gene was also induced following liver injury by CCl4 and in liver regeneration following partial hepatectomy. These results suggest that the D6.1A gene is related to stimulation of cell proliferation and differentiation status in C2 hepatoma cells [ABSTRACT FROM AUTHOR]

The nuclear-encoded fertility restorer locus for Ogura radish cytoplasmic male sterility (CMS) was previously transferred from radish into Brassica napus through intergeneric crosses, with the putative restorer gene(s) being expressed in leaves and flower buds. To identify transcripts corresponding to this restorer locus, we performed a PCR-selected cDNA subtraction using leaf mRNA from the restorer line and its recurrent parent. The resulting library was differentially screened using the subtracted cDNAs for the library itself and the cDNAs from the reverse subtraction (recurrent parent subtracted against the restorer line). Forty-eight cDNA clones were isolated based on their specific hybridization with cDNAs for the library construction. Sequence analyses revealed that these 48 clones correspond to only three different genes: two light-harvesting chlorophyll a/b-binding protein (Cab) genes and one chloroplast-encoded gene (a chloroplast 50S ribosomal protein gene). Of the two putative Cab genes, one showed polymorphism on the Southern blot with the EcoRI-digested genomic DNA from the restorer line and its recurrent parent. Segregation analyses confirmed that this putative Cab gene comes from radish DNA and co-segregates with the restorer locus. A sequence comparison of this putative Cab gene with its homeolog in Brassicanapus revealed that (1) their coding regions share a 94% similarity, (2) they differ in the size of the intron, and (3) they are identical in the deduced amino acid sequences, except that the one encoded at the restorer locus contained an additional alanine at position 34. [ABSTRACT FROM AUTHOR]

The shoot apical meristem (SAM) of Arabidopsis thaliana constitutes the tunica of L1 and L2 and the corpus represented by L3 cells. Regulatory networks involved in establishing and maintaining this structure of shoot meristems remain largely unknown. In order to identify the genes that function in the SAM, we performed cDNA subtraction experiments between wild-type and terminal flower 1 shoot apices. Here, we describe the cloning of a gene designated PDF1 (PROTODERMAL FACTOR1). In situ hybridization revealed that the expression of PDF1 is exclusively limited to the L1 layer of vegetative, inflorescence and floral meristems and to the protoderm of organ primordia. By contrast, PDF1 shows no detectable level of expression in the epidermis of mature organs. Specific expression of the PDF1 gene in protodermal cells is also observed during embryogenesis. The deduced amino acid sequence of PDF1 shares no significant homology with that of other known proteins but contains a putative signal peptide and novel proline-rich repeat motifs, suggesting a cell-wall protein. Possible roles of the PDF1 gene in the SAM are discussed. [ABSTRACT FROM AUTHOR]

We have constructed a soybean bacterial artificial chromosome (BAC) library using the plant introduction (PI) 437654. The library contains 73 728 clones stored in 192 384-well microtiter plates. A random sampling of 230 BACs indicated an average insert size of 136 kb with a range of 20 to 325 kb, and less than 4% of the clones do not contain inserts. Ninety percent of BAC clones in the library have an average insert size greater than 100 kb. Based on a genome size of 1115 Mb, library coverage is 9 haploid genome equivalents. Screening the BAC library colony filters with cpDNA sequences showed that contamination of the genomic library with chloroplast clones was low (1.85%). Library screening with three genomic RFLP probes linked to soybean cyst nematode (SCN) resistance genes resulted in an average of 18 hits per probe (range 7 to 30). Two separate pools of forward and reverse suppression subtractive cDNAs obtained from SCN-infected and uninfected roots of PI 437654 were hybridized to the BAC library filters. The 488 BACs identified from positive signals were fingerprinted and analyzed using FPC software (version 4.0) resulting in 85 different contigs. Contigs were grouped and analyzed in three categories: (1) contigs of BAC clones which hybridized to forward subtracted cDNAs, (2) contigs of BAC clones which hybridized to reverse subtracted cDNAs, and (3) contigs of BAC clones which hybridized to both forward and reverse subtracted cDNAs. This protocol provides an estimate of the number of genomic regions involved in early resistance response to a pathogenic attack. [ABSTRACT FROM AUTHOR]

To investigate the molecular mechanisms of implantation, we constructed a cDNA library of mouse uteri enriched with pregnancy-induced genes by subtractive hybridization and polymerase chain reaction (PCR). One of the isolated clones was the cDNA for the calcium binding protein D-9k (Cabp9k), which is considered to regulate intracytoplasmic concentration and transport of free calcium ions. Northern blot and in-situ hybridization analyses demonstrated that the Cabp9k mRNA was expressed in the endometrial epithelia, both luminal and glandular, in the uterus at the time of implantation. On pregnancy day 5 it was detected in the luminal, but not in the glandular, epithelia. In the oophorectomized adult mice, progesterone enhanced Cabp9k mRNA expression in the uterus, whereas oestrogen did not. Consistent with this, a nucleotide change was identified in the first intron of mouse Cabp9k gene corresponding to the oestrogen responsive element in the rat Cabp9k gene. Transfer of embryos into the uterine cavity of pseudopregnant mice reduced the expression of Cabp9k mRNA in the glandular epithelium, suggesting that Cabp9k mRNA expression is also regulated by embryonal signal(s). These findings demonstrated that Cabp9k mRNA is expressed in the endometrial epithelia during the implantation period under the control of progesterone and the presence of embryo, and suggest that CaBP9k plays a role in implantation by regulating the local calcium concentrations. [ABSTRACT FROM PUBLISHER]

We have used subtractive hybridization to isolate cDNA clones whose expression were up-regulated in transgenic tobacco ectopically expressing the rice homeobox gene, OSH1. Thirty-nine distinct cDNA clones, which we term HRGs (Homeobox Regulated Genes), were identified. Some of them were specifically expressed in transformants, indicating that their expression was possibly regulated by transgene. [ABSTRACT FROM AUTHOR]

Various genes are differentially expressed in cells during cell differentiation, development, aging, and in pathological conditions. To identify and isolate the genes that are specifically and differentially expressed in cells, we established a ligation-mediated polymerase chain reaction (PCR)-based method for cDNA subtraction. As this method is PCR-based, when target genes are expressed at high levels relative to the driver (a control pool for subtraction), even a small amount of target genes can be amplified. By this newly developed PCR-based subtraction method, a set of genes regulated by glucose were identified in a mouse insulinoma cell line. This PCR-based and non-radioactive subtraction method will be a powerful tool for identification of novel genes, specifically and differentally expressed in cells. [Diabetologia (1996) 39: 1293-1298] [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]