Application of cDNA array for studying the gene expression profile of mature appressoria of Magnaporthe grisea

Appressorium is an infection structure of the phytopathogenic fungus Magnaporthe grisea. Analysis of gene expression profiles ofappressorium development provides insight into the molecular basis of pathogenicity and control of this fungal plant disease. A cDNA array representing 2927 unique genes based on a large EST （expressed sequence tag） database ofM. grisea strain Y34 was constructed and used to profile the gene expression patterns at mycelium and appressorium maturation stages. Compared with mycelia, 55 up-regulated and 22 down-regulated genes were identified in mature appressoria. Among 77 genes, 16 genes showed no similarity to the genome sequences of M. grisea. A novel homologue of peptidyl-prolyl cis-trans isomerase was found to be expressed at low-level in mature appressoria of M. grisea. The results indicated that the genes such as pyruvate carboxylase, phospholipid metabolism-related protein and glyceraldehyde 3-phosphate dehydrogenase involved in gluconeogenesis, lipid metabolism and glycolysis, showed differential expression in mature appressoria. Furthermore, genes such as PTHll, beta subunit of G protein and SGTI involved in cell signalling, were expressed differentially in mature appressoria. Northern blot analysis was used to confirm the cDNA array results.

GENE EXPRESSION PROFILING OF PHENYLBUTYRATE INDUCED DIFFERENTIATION OF GLIOMA CELLS BY cDNA ARRAY

Objective: To analyze the changes of gene expression in phenylbutyrate induced differentiation of glioma cells. Methods: The expression levels of 14000 genes in glioma cells before and after inducement with sodium phenyl- butyrate for 2 h or 6 days were evaluated by cDNA array technique and proved by multi-dot blotting. Results: expression of 98 genes in glioma cells showed changes after the inducement. Some genes involved in transcription and translation and some oncogenes are down-regulated, while some gene involved in differentiation or apoptosis are up-regulated. 18 unknown expression sequencing tag (EST) changed too. Conclusion: A gene expression profile associated with differentiation of glioma cells was established.

Identification of genes differentially expressed in monocyte－deriveddendritic cells with 1α,25－dihydroxyvitamin D3 using cDNA arrays

In order to study the molecular mechanism of the inhibitory effect of 1,25-dihydroxyvitamin D3 on dendritic cells, experiments were performed using Atlas cDNA expression arrays from Clonetech to identify the differentially expressed genes of dendritic cells by 1,25-dihydroxyvitamin D3. Analysis of cDNA arrays revealed changes in the expression of 9 genes, including those involved in DNA binding and transcription, extracellular cell signaling and communication, intracellular transducers, as well as cell adhesions. The results indicated that a multiple molecular network is involved in the inhibitory role of 1,25-dihydroxyvitamin D3 on dendritic cells. The Atlas Array technology may facilitate the elucidation of complex pharmacological process of 1,25-dihydroxyvitamin D3 on dendritic cells.

Identification of brassinosteroid responsive genes in Arabidopsis by cDNA array

We have systematically monitored brassinosteroid (BR) responsive genes in a BR-deficient mutant det2 suspension culture of Arabidopsis by using a cDNA array approach. Among 13000 cDNA clones arrayed on filters, 53 BR responsive clones were identified and designated BRR1-BRR53. Sequence analysis of 43 clones showed that 19 clones are novel genes, 3 clones are genes involved in the control of cell division, 4 clones are genes related to plant stress responses, 4 clones are transcriptional factor or signal transduction component genes, and 3 clones are genes involved in RNA splicing or structure forming. In addition, we also found that BR regulated the transcription of genes related to many physiological processes, such as photoreaction, ion transportation and some metabolic processes. These findings present molecular evidence that BR plays an essential role in plant growth and development.

Identification of Novel Stress-responsive Transcription Factor Genes in Rice by cDNA Array Analysis

Numerous studies have shown that array of transcription factors has a role in regulating plant responses to environmental stresses. Only a small portion of them however, have been Identified or characterized. More than 2 300 putative transcription factors were predicted In the rice genome and more than half of them were supported by expressed sequences. With an attempt to Identify novel transcription factors involved in the stress responses, a cDNA array containing 753 putative rice transcription factors was generated to analyze the transcript profiles of these genes under drought and salinity stresses and absclsic acid treatment at seedling stage of rice. About 80% of these transcription factors showed detectable levels of transcript in seedling leaves. A total of 18 up-regulated transcription factors and 29 down-regulated transcription factors were detected with the folds of changes from 2.0 to 20.5 in at least one stress treatment. Most of these stress-responsive genes have not been reported and the expression patterns for five genes under stress conditions were further analyzed by RNA gel blot analysis. These novel stress-responsive transcription factors provide new opportunities to study the regulation of gene expression In plants under stress conditions.

Identification of Differentially Expressed Genes During Anther Abortion of Taigu Genic Male Sterile Wheat by Combining Suppression Subtractive Hybridization and cDNA Array

Talgu Genlc Male Sterile Wheat （TGMSW; Trltlcum aestlvum L.）, a dominant genic male sterile germplasm, is of considerable value in the genetic Improvement of wheat because of Its stable Inherence, complete male abortion, and high cross-fertilization rate. To Identify specially transcribed genes In sterile anther, a suppression subtractlve hybridization （aSH） library was constructed with sterile anther as the tester and fertile anther as the driver. A total of 2 304 SSH Inserts amplified by polymerase chain reaction were arrayed using robotic printing. The cDNA arrays were hybridized with 32P-labeled probes prepared from the RNA of forward- and reverse-subtracted anthers. Ninety-six clones were scored as upregulated in sterile anthers compared with the corresponding fertile anthers and some clones were selected for sequencing and analysis In GenBank. Based on their putative functions, 87 non-redundant clones were classified Into the following groups： （i） eight genes Involved In metabolic processes; （11） four material transportation genes; （iii） three signal transductlon-assoclated genes; （iv） four stress response and senescence-associated protein genes; （v） seven other functional protein genes; （vi） five genes with no known function; and （vii） another 56 genes with no match to the databases. To test the hybridization efficiency, eight genes were selected and analyzed by Northern blot. The results of the present study provide a comprehensive overview of the genes and gene products Involved In anther abortion In TGMSW.

POSSIBLE REASONS FOR TP53 ACCUMULATION IN NASO-PHARYNGEAL CARCINOMA USING ATLAS HUMAN CANCER cDNA EXPRESSION ARRAY

Objective: To compare gene expression profiles of nasopharyngeal carcinoma (NPC) tissue with that of control tissue by cDNA Array and to discuss possible reasons of TP53 accumulation in NPC tissue. Methods: (1) hybridization of Atlas Human Cancer cDNA Expression Array 7742-1; (2) analysis of Atlas Arrays using Atlasimage 1.01a; (3) verification of results of array by RT-PCR; (4) verification of protein expression alterations by immunohistochemistry. Results: (1) Of 588 tumor-related genes, 134 genes were upregulated, 88 downregulated; (2) Of 32 TP53-regulated genes, 13 genes were shown differential expression, 11 upregulated, 2 downregulated; (3) ATM and JNK2 were upregulated; (4) mRNA expression of ubiquitin-conjugating enzyme E2 (M74524) and ubiquitin-conjugating enzyme E2 (L22005) has no evident changes; Conclusion: (1) TP53 dysfunction exists in NPC tissues; (2) ATM and JNK might be the important causes of TP53 accumulation.

Global Identification of Significantly Expressed Genes in Developing Endosperm of Rice by Expression Sequence Tags and cDNA Array Approaches

Rice endosperm plays a very important role in seedling germination and determines the qualities of rice grain. Although studies on specific gene categories in endosperm have been carried out, global view of gene expression at a transcription level In rice endosperm Is still limited. To gain a better understanding of the global and tissue-specific gene expression profiles In rice endosperm, a cDNA library from rice endosperm of immature seeds was sequenced. A cDNA array was constructed based on the tentative unique transcripts derived from expression sequence tag （EST） assembling results and then hybridized with cDNAs from five different tissues or organs including endosperm, embryo, leaf, stem and root of rice. Significant redundancy was found for genes encoding prolamin, glutelin, allergen, and starch synthesis proteins, accounting for ~34% of the total ESTs obtained. The cDNA array revealed 87 significantly expressed genes In endosperm compared with the other four organs or tissues. These genes included 13 prolamin family proteins, 17 glutelin family proteins, 12 binding proteins, nine catalytic proteins and four ribosomal proteins, indicating a complicated biological processing in rice endosperm. In addition, Northern verification of 1,4-alpha-glucan branching enzyme detected two isoforms in rice endosperm, the larger one of which only existed in endosperm.

RAPID SCREENING OF AN ARRAYED cDNA LIBRARY BY IMPROVED PCR-BASED METHOD

The present study reports an improved PCR-based technique that allows quick and effective screening of cDNA libraries. First, the cDNA library was arrayed as follow: about 3 X 10’ cDNA clones were multiplied as individual plaques on solid medium in 24-well culture dishes at 1 200 plaque forming units per well. The phage suspension of each well was transferred to an individual microcentrifuge tube in 72-tube box. Then, box pool, row pools and column pools were set up that respectively represent a 72-tube box, rows and columns within the box. To screen a specific target cDNA,primers specific for novel ESTs ob- tained in our laboratory were employed to conduct PCR in a hierarchy mode. PCR began with the box pools, resulting in the identification of some Positive box pools. Then PCR went down to the row and col- umn pools of the positive box. The intersection of the positive row (s) and column (s) revealed the candi- date positive tubes. The specificity of PCR products were meanwhile checked by restriction enzyme diges- tion. Finally, hybridization was carried out to get single specific cDNA clomes from the positive tubes. This PCR-based technique features high specificity, high efficiency and less-cost in large-scale cDNA library screening. Our initial implementation of the technique resulted in the isolation of three longer different cD- NA clones from a human fetal brain cDNA library. Thus this improved technique can serve as an alterna-tive to the time-consuming and laborious conventional hybridization-based method for screening cDNA li-brary.

Blocking of N-acetylglucosaminyltransferase V induces cellular endoplasmic reticulum stress in human hepatocarcinoma 7721 cells

N-acetylglucosaminyltransferase V （GnT-V） is an important tumorigenesis and metastasis-associated enzyme. To study its biofunction, the GnT-V stably suppressed cell line （GnT-V-AS/7721） was constructed from 7721 hepatocarcinoma cells in previous study. In this study, cDNA array gene expression profiles were compared between GnT-V-AS/7721 and parental 7721 cells. The data indicated that GnT-V-AS/7721 showed a characteristic expression pattern consistent with the ER stress. The molecular mechanism of the ER stress was explored in GnT-V-AS/7721 by the analysis on key molecules in both two unfolded protein response （UPR） pathways. For ATF6 and Irel/XBP-1 pathway, it was evidenced by the up-regulation of BIP at mRNA and protein level, and the appearance of the spliced form ofXBP-1. As for PERK/eIF2α pathway, the activation of ER eIF2α kinase PERK was observed. To confirm the results from GriT-V-AS/7721 cells, the key molecules in the UPR were examined again in 7721 cells interfered with the GnT-V by the specific RNAi treatment. The results were similar with those from GnT-V-AS/7721, indicating that blocking of GnT-V can specifically activate ER stress in 7721 cells. Rate of 3H-Man incorporation corrected with rate of 3H-Leu incorporation in GnT-V-AS/7721 was down-regulated greatly compared with the control, which demonstrated the deficient function of the enzyme synthesizing N-glycans after GnT-V blocking. Moreover, the faster migrating form of chaperone GRP94 associated with the underglycosylation, and the extensively changed N-glycans structures of intracellular glycoproteins were also detected in GnT-V-AS/7721. These results supported the mechanism that blocking of GnT-V expression impaired functions of chaperones and N-glycan-synthesizing enzymes, which caused UPR in vivo.

Gene expression profile favoring phenotypic reversion:a clue for mechanism of tumor suppression by NF-IL6 3'UTR

Transfection of cDNA in 3'untranslated region of human nuclear factor for interleukin-6(NF-IL6 3'UTR)induced tumor suppression in a human hepatoma cell line.cDNA array analysis was used to reveal changes in gene expression profile leading to tumor suppression The results indicate that this suppression was not due to activation of dsRNA-dependent protein kinase,nor to inactivation ofoncogenes; rather,all the changes in expression of known genes,induced by NF-IL6 3'UTR cDNA may be ascribed to the suppression of cellular malignancy.Therefore,our results imply that this 3'untranslated region may have played role of a regulator of gene expression profile.

Gene expression profiling defined pathways correlated with fibroblast cell proliferation induced by Opisthorchis viverrini excretory /secretory product

AIM： To investigate the mechanism of fibroblast cell proliferation stimulated by the Opisthorchis viverrini excretory/secretory （ES） product. METHODS： NIH-3T3, mouse fibroblast cells were treated with O. viverrini ES product by non-contact co-cultured with the adult parasites. Total RNA from NIH-3T3 treated and untreated with O. viverrini was extracted, reverse transcribed and hybridized with the mouse 15K complementary DNA （cDNA） array. The result was analyzed by ArrayVision version 5 and GeneSpring version 5 softwares. After normalization, the ratios of gene expression of parasite treated to untreated NIH-3T3 cells of 2-and more-fold upregulated was defined as the differentially expressed genes. The expression levels of the signal transduction genes were validated by semiquantitative SYBR-based real-time RT-PCR. RESULTS： Among a total of 15 000 genes/ESTs, 239 genes with established cell proliferation-related function were 2 fold-and more-up-regulated by O. viverrini ES product compared to those in cells without exposure to the parasitic product. These genes were classified into groups including energy and metabolism, signal transduction, protein synthesis and translation, matrix and structural protein, transcription control, cell cycle and DNA replication. Moreover, the expressions of serinethreonine kinase receptor, receptor tyrosine kinase and collagen production-related genes were up-regulated by O.viverrini ES product, The expression level of signal transduction genes, pkC, pdgfra, jak 1, eps 8, tgfβ 1/4,strap and h ras measured by real-time RT-PCR confirmed their expression levels to those obtained from cDNA array. However, only the up-regulated expression of pkC,eps 8 and tgfβ3 1/4 which are the downstream signaling molecules of either epidermal growth factor （EGF） or transforming growth factor-β （TGF-β） showed statistical significance （P 〈 0.05）.CONCLUSION： O. viverrini ES product stimulates the significant changes of gene expression in several functional categories and these mainly include transcripts related to cell proliferation. The TGF-β and EGF signal transduction pathways are indicated as the possible pathways of O. viverrini-driven cell proliferation.

Porous Silicon as a Carrier of Sensing Materials in Sensors

Novel potassium ion selective electrodes (K^+ISEs) and cDNA array sensors based on porous silicon (PS) have been developed.The calibration curve for the K^+ISEs is linear within a wide range of pK=2.0～6.0 with the slope of 56 mV per decade,which is near Nernst response.The response time and detection limit are within 31 s and 0.5μmol/L,respectively.The selective coefficient for Na^+ is-3.8,satisfies the requirement for the assay of blood potassium.The response variation is within 2 mV during 2 months.The binding capacity,the dynamic range and the detection limit of the DNA sensors were improved by replacing glass slide with PS substrates.The cDNA array sensors can bear 80℃of high temperature,75% of humidity,3.6 kLx of irradiation and keep stable within 10 days when they are exposed in air.Good performances of the K^+ISE and the cDNA array sensor are attributed to the large internal surface area and the easily modified microstructure of PS.

TNF-alpha-induced metastasis gene changes in MCF-7 cells

Objective： Studies have shown that TNF- α secreted by tumor ceils and macrophages infiltrated into the tumor microenvironment might promote the metastasis of a variety of malignant cancers, including breast cancer. The present study was designed to detect global metastasis-related gene expression changes of MCF-7 cells treated by low dose TNF-α and to further explore the mechanisms by which TNF- α contributes to metastasis. Methods： MCF-7 cells were cultured and treated with low dose TNF-α （20 ng/ml）, cDNA array analysis was applied to detect the metastasis related gene expressions. Results： A total of 36 gene expressions were significantly regulated by TNF- α. Functional analysis indicates that the altered genes belong to different functional group. Most of the genes changed may promote the metastasis of MCF-7 cells while the others may inhibit metastasis. The changes observed in gene expression following TNF-α were somewhat time dependent. Conclusion： TNF-α can enhance the invasive ability of MCF-7 cells, partly by regulating a series of metastasis related genes, and these genes may take part in every step of metastasis. Some of the genes deserve further study.