Oligonucleotide DNA Microarray Research Articles

マイクロアレイを用いた着色期ブドウ果皮における光・低温誘導性遺伝子の探索

Flavonoid biosynthesis in grape (Vitis spp.) berry skin is affected by environmental factors such as light and temperature. However, the components of the light signaling and low-temperature-induced abscisic acid (ABA) signaling networks related to flavonoid accumulation in grape berry skin have not been fully elucidated, and how environmental conditions affect these components remains unclear. To clarify the details of the possible light- and ABA-related signal transduction networks, we developed a grape oligo-DNA microarray (38,549 independent probes) using the publicly available genomic sequence of grape, and performed comprehensive transcriptome analysis using detached ‘Pione’ grape (Vitis labruscana × V. vinifera) berries cultured under different light and temperature conditions. Using the microarray data, we explored the light-inducible and low-temperature-inducible genes in post-veraison grape berries. We identified 40 light-inducible genes, 55 low-temperature-inducible genes, and 34 genes induced by light plus low temperature. Among these, we selected elongated hypocotyl 5 (HY5), open stomata 1 (OST1), and enhanced response to ABA 1 (ERA1) as candidate light-inducible, low-temperature-inducible, and light- plus low-temperature-inducible genes, respectively. We investigated their detailed expression characteristics in grape accessions by means of quantitative real-time polymerase chain reaction analyses, and hypothesized that HY5, OST1, and ERA1 might be involved in flavonoid biosynthesis via light signaling and low-temperature signaling networks. We also established an extensive catalog of gene expression patterns to support future investigations of other candidate genes that respond to light and temperature in grape berry skin.

Human blood monocytes support persistence, but not replication of the intracellular pathogen C. pneumoniae.

BackgroundIntracellular pathogens have devised various mechanisms to subvert the host immune response in order to survive and replicate in host cells. Here, we studied the infection of human blood monocytes with the intracellular pathogen C. pneumoniae and the effect on cytokine and chemokine profiles in comparison to stimulation with LPS.ResultsMonocytes purified from peripheral blood mononuclear cells by negative depletion were infected with C. pneumoniae. While immunofluorescence confirmed the presence of chlamydial lipopolysaccharide (LPS) in the cytoplasm of infected monocytes, real-time PCR did not provide evidence for replication of the intracellular pathogen. Complementary to PCR, C. pneumoniae infection was confirmed by an oligonucleotide DNA microarray for the detection of intracellular pathogens. Raman microspectroscopy revealed different molecular fingerprints for infected and non-infected monocytes, which were mainly due to changes in lipid and fatty acid content. Stimulation of monocytes with C. pneumoniae or with LPS induced similar profiles of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6, but higher levels of IL-1β, IL-12p40 and IL-12p70 for C. pneumoniae which were statistically significant. C. pneumoniae also induced release of the chemokines MCP-1, MIP-1α and MIP-1β, and CXCL-8, which correlated with TNF-α secretion.ConclusionInfection of human blood monocytes with intracellular pathogens triggers altered cytokine and chemokine pattern as compared to stimulation with extracellular ligands such as LPS. Complementing conventional methods, an oligonucleotide DNA microarray for the detection of intracellular pathogens as well as Raman microspectroscopy provide useful tools to trace monocyte infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-014-0060-1) contains supplementary material, which is available to authorized users.

Gene expression profiles in the bovine corpus luteum (CL) during the estrous cycle and pregnancy: possible roles of chemokines in regulating CL function during pregnancy.

To determine functional differences between the corpus luteum (CL) of the estrous cycle and pregnancy in cows, gene expression profiles were compared using a 15 K bovine oligo DNA microarray. In the pregnant CL at days 20–25, 40–45 and 150–160, the expressions of 138, 265 and 455 genes differed by a factor of > 2-fold (P < 0.05) from their expressions in the cyclic CL (days 10–12 of the estrous cycle). Messenger RNA expressions of chemokines (eotaxin, lymphotactin and ENA-78) and their receptors (CCR3, XCR1 and CXCR2) were validated by quantitative real-time PCR. Transcripts of eotaxin were more abundant in the CL at days 40–45 and 150–160 of pregnancy than in the cyclic CL (P < 0.01). In contrast, the mRNA expressions of lymphotactin, ENA-78 and XCR1 were lower in the CL of pregnancy (P < 0.05). Messenger RNAs of CCR3 and CXCR2 were similarly detected both in the cyclic and pregnant CL. Tissue protein levels of eotaxin were significantly higher in the CL at days 150–160 of pregnancy than in the CL at other stages, whereas the lymphotactin protein levels in the CL at days 20–25 of pregnancy were lower (P < 0.05). Immunohistochemical staining showed that CCR3 was expressed in the luteal cells and that XCR1 was expressed in both the luteal cells and endothelial cells. Collectively, the different gene expression profiles may contribute to functional differences between the cyclic and pregnant CL, and chemokines including eotaxin and lymphotactin may regulate CL function during pregnancy in cows.

Cell-type specific transcriptomic profiling to dissect mechanisms of differential dendritogenesis.

The establishment, maintenance and modulation of cell-type specific neural architectures are critically important to the formation of functional neural networks. At the neuroanatomical level, differential patterns of dendritic arborization directly impact neural function and connectivity, however the molecular mechanisms underlying the specification of distinct dendrite morphologies remain incompletely understood. To address this question, we analyzed global gene expression from purified populations of wild-type class I and class IV Drosophila melanogaster dendritic arborization (da) sensory neurons compared to wild-type whole larval RNA using oligo DNA microarray expression profiling. Herein we present detailed experimental methods and bioinformatic analyses to correspond with our data reported in the Gene Expression Omnibus under accession number GSE46154. We further provide R code to facilitate data accession, perform quality controls, and conduct bioinformatic analyses relevant to this dataset. Our cell-type specific gene expression datasets provide a valuable resource for guiding further investigations designed to explore the molecular mechanisms underlying differential patterns of neuronal patterning.

P2-25-5 - Efficacy of Nab-Paclitaxel on 5-Fluorouracil (5Fu) Resistant Human Gastric Cancer Cell Lines

Purpose: 5-Fluorouracil (5FU)-based combination chemotherapy is widely used for the treatment of advanced gastric cancer in Japan. Thus, the development of effective chemotherapy for 5FU-resistant gastric cancer after 5FU-based chemotherapy is important. Nab-paclitaxel (Nab-PTX) is an albumin-bound, 130nm particle form of paclitaxel that exhibits a higher activity and lower toxicity than solvent-based paclitaxel. In the present study, the activity of Nab-PTX alone or in combination with 5FU was evaluated in four 5FU resistant cell lines.Method: 5FU-resistant human gastric cancer cell lines were established from parent cells (MKN-45, MKN-74, NCI-N87, and KATO-III) by continuous exposure to 5FU for over a one-year period. The sensitivities of these cell lines to 5FU and Nab-PTX were evaluated using the WST-8 colorimetric assay. The effects of the combination of 5FU and Nab-PTX was evaluated by the combination index (CI) value. The gene expressions of secreted protein acidic and rich in cysteine (SPARC) was evaluated using a Whole Human Genome 44K oligo DNA microarray of Agilent Technologies.Results: The IC50 value of 5FU ranged from 2.35 to 36.7 µM, and its ratio (resistant/parent) ranged from 2.6 to 15.6. The IC50 value of Nab-PTX ranged from 4.45 to 54.5 nM, and its ratio ranged from 1.0 to 1.2. Therefore, Nab-PTX showed no signs of cross-resistance to 5FU. The calculated CI value treated with 5FU and Nab-PTX was approximately 1; therefore, Nab-PTX additively inhibited the cell growth in combination with 5FU. Interestingly, the combination of 5FU and Nab-PTX had an additive activity in the 5FU-resistant cell lines. The relative expression of SPARC mRNA in the resistant cell lines to that in the parent ones ranged from 0.86 to 1.47, and SPARC expression might be weakly correlated with the acquisition of 5FU resistance.Conclusion: Nab-PTX might be a promising agent for the treatment of 5FU-resistant human gastric cancer when used alone or in combination with 5FU.

Loss of progesterone receptor links to high proliferation and increases from primary to metastatic endometrial cancer lesions

ObjectiveIn endometrial cancer loss of progesterone receptor (PR, gene name PGR) is associated with aggressive disease and altered response to hormonal treatment. The aim of this study was to investigate changes in PR expression level with disease progression, and explore whether differences in gene expression according to PR status can be linked to processes involved in cancer development elucidating new therapeutic opportunities. Methods686 primary endometrial cancers and 171 metastatic lesions were investigated for PR expression in relation to clinical and histopathological data. Protein levels were investigated by immunohistochemistry and reverse phase protein array, and mRNA levels by DNA oligonucleotide microarray. ResultsPR protein level was significantly associated with PGR mRNA expression (P<0.001) and patient survival (P<0.001). Loss of PR increased with disease progression, with 23% of the primary tumours and 76% of metastases demonstrating PR loss. Using a cell cycle progression signature score, PR loss was associated with increased proliferation for both oestrogen receptor (ER) positive and negative tumours. Through a Connectivity Map search, CDK inhibitors and other drugs with anti-proliferative effects were suggested in particular for treatment of patients with loss of PR. ConclusionLoss of PR in endometrial cancer is associated with increased proliferation, poor survival, and increases from primary to metastatic lesions. Based on expression profiles, CDK inhibitors may have activity in PR negative tumours, supporting further testing in clinical trials for patients with systemic endometrial cancer dependent on PR status.

Abstract 777: Efficacy of nab-paclitaxel on 5-fluorouracil (5-FU) resistant human gastric cancer cell lines

Abstract Purpose: 5-Fluorouracil (5-FU)-based combination chemotherapy is widely used for the treatment of advanced gastric cancer in Japan. Thus, the development of effective chemotherapy for 5-FU-resistant gastric cancer after 5-FU-based chemotherapy is important. Nab-paclitaxel is an albumin-bound, 130-nm particle form of paclitaxel that exhibits a higher activity and lower toxicity than solvent-based paclitaxel. In the present study, the activity of nab-paclitaxel alone or in combination with 5-FU was evaluated in four 5-FU resistant cell lines. Method: 5-FU-resistant human gastric cancer cell lines were established from parent cells (MKN-45, MKN-74, NCI-N87, and KATO-III) by continuous exposure to escalating concentrations of 5-FU over a one-year period. The sensitivities of these cell lines to chemotherapeutic agents (5-FU and nab-paclitaxel) were evaluated using the WST-8 colorimetric assay. To evaluate the effects of the combination of 5-FU and nab-paclitaxel, the combination index (CI) value was calculated using CalcuSyn 2.0 (Biosoft, Cambridge, UK). The gene expressions of secreted protein acidic and rich in cysteine (SPARC) in the parent and 5-FU-resistant cell lines were evaluated using a Whole Human Genome 44K oligo DNA microarray (Agilent Technologies, Santa Clara, CA). Results: The IC50 value of 5-FU ranged from 2.35 to 36.7 μM, and its ratio (resistant cell lines/parent cell lines) ranged from 2.6 to 15.6. The IC50 value of nab-paclitaxel ranged from 4.45 to 54.5 nM, and its ratio ranged from 1.0 to 1.2. Therefore, the 5-FU-resistant cell lines used in this study showed no signs of cross-resistance to nab-paclitaxel. The calculated CI value treated with 5-FU and nab-paclitaxel was approximately 1; therefore, nab-paclitaxel additively inhibited the cell growth when administered in combination with 5-FU. Interestingly, the combination of 5-FU and nab-paclitaxel had an additive cytotoxic activity in the 5-FU-resistant human gastric cell lines in vitro. The relative expression of SPARC mRNA in the resistant cell lines to that in the parent ones ranged from 0.86 to 1.47, and SPARC expression might be weakly correlated with the acquisition of 5-FU resistance. Conclusion: Nab-paclitaxel might be a promising agent for the treatment of 5-FU-resistant human gastric cancer when used alone or in combination with 5-FU. These results suggest that nab-paclitaxel might be a promising chemotherapy for gastric cancer patients who relapse after undergoing 5-FU-based chemotherapy. Citation Format: Mamoru Nukatsuka, Takashi Kobunai, Kazuaki Matsuoka, Teiji Takechi. Efficacy of nab-paclitaxel on 5-fluorouracil (5-FU) resistant human gastric cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 777. doi:10.1158/1538-7445.AM2014-777

대장암 세포주에서 주박 추출물의 유기용매 분획물의 항성장 활성

In the present study, we prepared eighty-five different kinds of lees extracts and their solvent fractions and investigated their anti-proliferative activities against human colorectal cancer HCT116 cells. HCT116 cells were treated with eighty-five solvent fractions of lees extracts and then cell viability was measured using MTS assay. Among the treated solvent fractions, three solvent fractions (KSD-E1-3, KSD-E2-3, and KSD-E4-3) were selected based on cell viability assay. In addition, we performed an oligo DNA microarray analysis to analyze the gene expression changes by treatment of KSD-E1-3 in HCT116 cells. Among the up-regulated genes, we selected 4 genes (NAG-1, ATF3, p21, and DDIT3) and performed RT-PCR using gene-specific primers. Among the treated solvent fractions, KSD-E1-3 dramatically induced the expressions of the four selected genes. In addition, we investigated whether the up-regulations of those genes were dependent on the transcription factor p53’s presence using p53 null HCT116 cells. The results indicate that the up-regulations of NAG-1, ATF3, and DDIT3 are not dependent on the p53 presence, whereas p21 is dependent on the p53 presence. These findings may help to understand the molecular mechanisms of the anti-proliferative activity mediated by rice wine lees in human colorectal cancer cells.

주박 추출물과 이들의 유기용매 분획물에 의한 항염증 활성

본 연구에서는 전통주 제조시 부산물로 생산되는 주박과 누룩으로부터 추출물과 유기용매 분획물을 총 85종을 제조하고, 이들에 의한 항염증 활성을 연구하였다. 85종의 분획물 중 선별한 세 가지의 분획물(KSD-E1-3, KSD-E2-3, KSD-E4-3)에 의해서 LPS에 의해 염증이 유도된 RAW 264.7 세포주에서 nitric oxide 생산이 현저히 감소됨을 확인하였다. 또한, 세가지 분획물에 의해 염증유발 유전자인 COX-2, TNF-alpha, 그리고 iNOS 유전자의 발현이 감소되었다. 세 가지 분획물 중 KSD-E4-3에 의한 항염증 활성의 작용기전을 이해하기 위하여 oligo DNA microarray를 수행하였다. 마이크로어레이 결과 발현이 감소된 유전자 중 염증과 관련된 유전자 6개(IL-1F6, iNOS, IL-10, Fabp4, IL-1RN, CSF2)를 선택하여, RT-PCR과 정량적 real-time PCR을 수행하였다. 그 결과, 모든 유전자의 발현이 감소됨을 확인하였다. 결론적으로, 이러한 연구결과는 전통주 주박이 항염증 활성을 가지고 있는 식품이나 약품을 개발하는데 필요한 새로운 자원으로서 활용 가능함을 시사하는 것이다. In the current study, we prepared eighty-five different kinds of solvent fractions of rice wine lees and nuruk extracts and investigated their effects on cell viability and nitric oxide (NO) production in mouse RAW 264.7 cells. Among the treated solvent fractions, only three solvent fractions (KSD-E1-3, KSD-E2-3 and KSD-E4-3) significantly decreased NO production in LPS-activated RAW 264.7 cells without affecting cell viability. And, they also reduced the expression of pro-inflammatory genes such as COX-2, TNF-alpha and iNOS. To understand the molecular mechanisms involved in the inhibition of inflammation in (KSD-E4-3)-treated RAW 264.7 cells, we carried out oligo DNA microarray analysis using Agilent Mouse microarray. To confirm microarray data, 6 genes (IL-1F6, iNOS, IL-10, Fabp4, IL-1RN and CSF2) were selected and performed RT-PCR and quantitative real-time PCR analysis with gene specific primers. The results of RT-PCR and real-time PCR agreed with microarray data. Overall, our results suggest that rice wine lees can be a novel resource for the development of foods and drugs which possess anti-inflammatory activity.

Mapping in an apple (Malus x domestica) F1segregating population based on physical clustering of differentially expressed genes

BackgroundApple tree breeding is slow and difficult due to long generation times, self-incompatibility, and complex genetics. The identification of molecular markers linked to traits of interest is a way to expedite the breeding process. In the present study, we aimed to identify genes whose steady-state transcript abundance was associated with inheritance of specific traits segregating in an apple (Malus × domestica) rootstock F1 breeding population, including resistance to powdery mildew (Podosphaera leucotricha) disease and woolly apple aphid (Eriosoma lanigerum).ResultsTranscription profiling was performed for 48 individual F1 apple trees from a cross of two highly heterozygous parents, using RNA isolated from healthy, actively-growing shoot tips and a custom apple DNA oligonucleotide microarray representing 26,000 unique transcripts. Genome-wide expression profiles were not clear indicators of powdery mildew or woolly apple aphid resistance phenotype. However, standard differential gene expression analysis between phenotypic groups of trees revealed relatively small sets of genes with trait-associated expression levels. For example, thirty genes were identified that were differentially expressed between trees resistant and susceptible to powdery mildew. Interestingly, the genes encoding twenty-four of these transcripts were physically clustered on chromosome 12. Similarly, seven genes were identified that were differentially expressed between trees resistant and susceptible to woolly apple aphid, and the genes encoding five of these transcripts were also clustered, this time on chromosome 17. In each case, the gene clusters were in the vicinity of previously identified major quantitative trait loci for the corresponding trait. Similar results were obtained for a series of molecular traits. Several of the differentially expressed genes were used to develop DNA polymorphism markers linked to powdery mildew disease and woolly apple aphid resistance.ConclusionsGene expression profiling and trait-associated transcript analysis using an apple F1 population readily identified genes physically linked to powdery mildew disease resistance and woolly apple aphid resistance loci. This result was especially useful in apple, where extreme levels of heterozygosity make the development of reliable DNA markers quite difficult. The results suggest that this approach could prove effective in crops with complicated genetics, or for which few genomic information resources are available.

Transcriptome analysis of barley identifies heat shock and HD-Zip I transcription factors up-regulated in response to multiple abiotic stresses

Analyzing barley gene expression profiles in response to abiotic stress is critical to understanding how barley manages stress, and provides vital information to improve environmental stress tolerance for stable crop production. We developed an Agilent 60-mer oligo DNA microarray with 42,491 probe sets based on the sequences of 36,632 barley (Hordeum vulgare L.) full-length cDNA clones and conducted global expression profiling on barley seedlings subjected to desiccation, salt, cold and abscisic acid (ABA). We identified 281 genes that were differentially expressed in response to desiccation, salt, and cold stresses and ABA treatment. Among them, a class C heat shock transcription factor (HvHsfC1) and a homeodomain leucine zipper (HD-Zip) family I transcription factor (HvHox22) showed more than tenfold and fourfold higher expression, respectively, in response to the stimuli. Heat shock and HD-Zip transcription factors function as important regulators in stress responses in rice (Oryza sativa) and Arabidopsis thaliana; our results suggest these two transcription factors also play important roles in abiotic stress responses in barley. We mapped HvHox22 to the long arm of chromosome 2H and HvHsfC1 to the long arm of 4H, where drought resistance quantitative trait loci were previously detected. Our microarray data and identification of these stress response genes provide key information for dissection of the mechanism of abiotic stress tolerance in barley.

Expression of proteins associated with adipocyte lipolysis was significantly changed in the adipose tissues of the obese spontaneously hypertensive/NDmcr-cp rat

BackgroundThe etiology of the metabolic syndrome is complex, and is determined by the interplay of both genetic and environmental factors. The present study was designed to identify genes and proteins in the adipose tissues with altered expression in the spontaneously hypertensive/NIH –corpulent rat, SHR/NDmcr-cp (CP) and to find possible molecular targets associated with the pathogenesis or progression of obesity related to the metabolic syndrome.MethodsWe extracted RNAs and proteins from the epididymal adipose tissues in CP, SHR/Lean (Lean), and Wistar Kyoto (WKY) rats and performed microarray analysis and two-dimensional difference in gel electrophoresis (2D-DIGE) linked to a matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS).ResultsThe results showed different mRNA and protein expression levels in the adipose tissue: oligo DNA microarray identified 33 genes that were significantly (P < 0.01) up-regulated and 17 genes significantly down-regulated in CP compared with WKY and Lean rats at both 6 and 25 weeks of age. The affected genes-proteins were associated with lipolytic enzymes stimulated by peroxisome proliferator-activated receptor (PPAR) signaling. Further analysis using the 2D-DIGE connected with MALDI-TOF/TOF analysis, the expression of monoglyceride lipase (MGLL) was significantly up-regulated and that of carboxylesterase 3 (CES3) was significantly down-regulated in 6- and 25-week-old CP compared with age-matched control (WKY and Lean rats).ConclusionsOur results suggest the possible involvement of proteins associated with adipocyte lipolysis in obesity related to the metabolic syndrome.

Gene expression profiling of mesenteric lymph nodes from sheep with natural scrapie

BackgroundPrion diseases are characterized by the accumulation of the pathogenic PrPSc protein, mainly in the brain and the lymphoreticular system. Although prions multiply/accumulate in the lymph nodes without any detectable pathology, transcriptional changes in this tissue may reflect biological processes that contribute to the molecular pathogenesis of prion diseases. Little is known about the molecular processes that occur in the lymphoreticular system in early and late stages of prion disease. We performed a microarray-based study to identify genes that are differentially expressed at different disease stages in the mesenteric lymph node of sheep naturally infected with scrapie. Oligo DNA microarrays were used to identify gene-expression profiles in the early/middle (preclinical) and late (clinical) stages of the disease.ResultsIn the clinical stage of the disease, we detected 105 genes that were differentially expressed (≥2-fold change in expression). Of these, 43 were upregulated and 62 downregulated as compared with age-matched negative controls. Fewer genes (50) were differentially expressed in the preclinical stage of the disease. Gene Ontology enrichment analysis revealed that the differentially expressed genes were largely associated with the following terms: glycoprotein, extracellular region, disulfide bond, cell cycle and extracellular matrix. Moreover, some of the annotated genes could be grouped into 3 specific signaling pathways: focal adhesion, PPAR signaling and ECM-receptor interaction. We discuss the relationship between the observed gene expression profiles and PrPSc deposition and the potential involvement in the pathogenesis of scrapie of 7 specific differentially expressed genes whose expression levels were confirmed by real time-PCR.ConclusionsThe present findings identify new genes that may be involved in the pathogenesis of natural scrapie infection in the lymphoreticular system, and confirm previous reports describing scrapie-induced alterations in the expression of genes involved in protein misfolding, angiogenesis and the oxidative stress response. Further studies will be necessary to determine the role of these genes in prion replication, dissemination and in the response of the organism to this disease.

The Prevalence of Chromosomal Deletions Relating to Developmental Delay and/or Intellectual Disability in Human Euploid Blastocysts

Chromosomal anomalies in human embryos produced by in vitro fertilization are very common, which include numerical (aneuploidy) and structural (deletion, duplication or others) anomalies. Our previous study indicated that chromosomal deletion(s) is the most common structural anomaly accounting for approximately 8% of euploid blastocysts. It is still unknown if these deletions in human euploid blastocysts have clinical significance. In this study, we analyzed 15 previously diagnosed euploid blastocysts that had chromosomal deletion(s) using Agilent oligonucleotide DNA microarray platform and localized the gene location in each deletion. Then, we used OMIM gene map and phenotype database to investigate if these deletions are related with some important genes that cause genetic diseases, especially developmental delay or intellectual disability. As results, we found that the detectable chromosomal deletion size with Agilent microarray is above 2.38 Mb, while the deletions observed in human blastocysts are between 11.6 to 103 Mb. With OMIM gene map and phenotype database information, we found that deletions can result in loss of 81-464 genes. Out of these genes, 34–149 genes are related with known genetic problems. Furthermore, we found that 5 out of 15 samples lost genes in the deleted region, which were related to developmental delay and/or intellectual disability. In conclusion, our data indicates that all human euploid blastocysts with chromosomal deletion(s) are abnormal and transfer of these embryos may cause birth defects and/or developmental and intellectual disabilities. Therefore, the embryos with chromosomal deletion revealed by DNA microarray should not be transferred to the patients, or further gene map and/or phenotype seeking is necessary before making a final decision.

Custom Microarray Analysis for Transcript Profiling of Dormant Vegetative Buds of Japanese Apricot during Prolonged Chilling Exposure

Bud dormancy is a critical developmental process for perennial plant survival, and also an important physiological phase that affects the next season’s growth of temperate fruit trees. Bud dormancy is regulated by multiple genetic factors, and affected by various environmental factors, tree age and vigor. To understand the molecular mechanism of bud dormancy in Japanese apricot (Prunus mume Sieb. et Zucc.), we constructed a custom oligo DNA microarray covering the Japanese apricot dormant bud ESTs referring to the peach (P. persica) genome sequence. Because endodormancy release is a chilling temperature-dependent physiological event, genes showing chilling-mediated differential expression patterns are candidates to control endodormancy release. Using the microarray constructed in this study, we monitored gene expression changes of dormant vegetative buds of Japanese apricot during prolonged artificial chilling exposure. In addition, we analyzed seasonal gene expression changes. Among the 58539 different unigene probes, 2345 and 1059 genes were identified as being more than twofold up-regulated and down-regulated, respectively, following chilling exposure for 60 days (P < 0.05). Cluster analysis suggested that the expression of the genes showing expression changes by artificial chilling exposure were coordinately regulated by seasonal changes. The down-regulated genes included P. mume DORMANCY-ASSOCIATED MADS-box genes, which supported previous quantitative RT-PCR and EST analyses showing that these genes are repressed by prolonged chilling exposure. The genes encoding lipoxygenase were markedly up-regulated by prolonged chilling. Our parametric analysis of gene-set enrichment suggested that genes related to jasmonic acid (JA) and oxylipin biosynthesis and metabolic processes were significantly up-regulated by prolonged chilling, whereas genes related to circadian rhythm were significantly down-regulated. The results obtained from microarray analyses were verified by quantitative RT-PCR analysis of selected genes. Taken together, we have concluded that the microarray platform constructed in this study is applicable for deeper understanding of the molecular network related to agronomically important bud physiology, including dormancy release.

Changes in genome-wide gene expression during allopolyploidization and genome stabilization in hexaploid wheat.

Allopolyploidization is an important evolutionary event in plants, but its genome-wide effects are not fully understood. Common wheat, Triticum aestivum (AABBDD), evolved through amphidiploidization between T. turgidum (AABB) and Aegilops tauschii (DD). Here, global gene expression patterns in the seedlings of a synthetic triploid wheat line (ABD), its chromosome-doubled hexaploid (AABBDD) and stable synthetic hexaploid (AABBDD), and the parental lines T. turgidum (AABB) and Ae. tauschii (DD) were compared using an oligo-DNA microarray to identify metabolic pathways affected by the genome conflict that occurs during allopolyploidization and genome stabilization. Characteristic gene expression patterns of non-additively expressed genes were detected in the newly synthesized triploid and hexaploid, and in the stable synthetic hexaploid. Hierarchical clustering of all differentially expressed and non-additively expressed genes revealed that the gene expression patterns of the triploid (ABD) were similar to those of the maternal parent (AABB), and that expression patterns in successive generations arising from self-pollination became closer to that of the pollen parent (DD). The non-additive gene expression profiles markedly differed between the triploid (ABD) and chromosome-doubled hexaploid (AABBDD), as supported by Gene Ontology (GOSlim) analysis. Four hundred and nineteen non-additively expressed genes were commonly detected in all three generations. GOSlim analysis indicated that these non-additively expressed genes were predominantly involved in "biological pathways". Notably, four of 11 genes related to sugar metabolism displayed elevated expression throughout allopolyploidization. These may be useful candidates for promoting heterosis and adaptation in plants.

PhyloFlu, a DNA Microarray for Determining the Phylogenetic Origin of Influenza A Virus Gene Segments and the Genomic Fingerprint of Viral Strains

Recent evidence suggests that most influenza A virus gene segments can contribute to the pathogenicity of the virus. In this regard, the hemagglutinin (HA) subtype of the circulating strains has been closely surveyed, but the reassortment of internal gene segments is usually not monitored as a potential source of an increased pathogenicity. In this work, an oligonucleotide DNA microarray (PhyloFlu) designed to determine the phylogenetic origins of the eight segments of the influenza virus genome was constructed and validated. Clades were defined for each segment and also for the 16 HA and 9 neuraminidase (NA) subtypes. Viral genetic material was amplified by reverse transcription-PCR (RT-PCR) with primers specific to the conserved 5' and 3' ends of the influenza A virus genes, followed by PCR amplification with random primers and Cy3 labeling. The microarray unambiguously determined the clades for all eight influenza virus genes in 74% (28/38) of the samples. The microarray was validated with reference strains from different animal origins, as well as from human, swine, and avian viruses from field or clinical samples. In most cases, the phylogenetic clade of each segment defined its animal host of origin. The genomic fingerprint deduced by the combined information of the individual clades allowed for the determination of the time and place that strains with the same genomic pattern were previously reported. PhyloFlu is useful for characterizing and surveying the genetic diversity and variation of animal viruses circulating in different environmental niches and for obtaining a more detailed surveillance and follow up of reassortant events that can potentially modify virus pathogenicity.

Mosaic pregnancy after transfer of a “euploid” blastocyst screened by DNA microarray

BackgroundHigh proportions of human embryos produced by in vitro fertilization are aneuploidy and mosaic. DNA microarray is one of the most practical screening methods to select euploid embryos for transfer. However, mosaic pregnancy is still possible due to embryonic mosacism. Here we report a successful pregnancy after transfer of a mosaic blastocyst with euploid inner cell mass.MethodsA woman with a previous trisomy 13 pregnancy pursued infertility treatment with preimplantation genetic screening by a trophectoderm biopsy and DNA microarray. NimbleGen oligonucleotide DNA microarray was applied to biopsied samples from 13 blastocysts. A euploid blastocyst was transferred to the patient and subsequent prenatal cytogenetic tests were performed by FISH and/or G banding.ResultsFollowing DNA microarray, it was found that 5 blastocysts were euploid and 8 were aneuploidy. Transfer of one euploid blastocyst resulted in a clinical pregnancy. Prenatal cytogenetic tests of samples biopsied from chorionic villi sample showed both trisomy 21 (47 XX, +21) and euploid (46, XX) cells. Further prenatal cytogenetic test with a sample from amniotic fluid indicated that all cells were euploid (46, XX). The pregnancy was continued and a healthy girl was delivered after 41 weeks of gestation.ConclusionsThis is the first report to indicate a mosaic pregnancy after transfer of a “euploid” blastocyst that was screened by DNA microarray, and the case further confirms that mosaicism is present in human blastocysts produced by in vitro fertilization.

Gene expression in the vascular wall of the aortic arch in spontaneously hypertensive hyperlipidemic model rats using DNA microarray analysis

AimsIn recent years, there has been an increase in patients with arteriosclerosis and the risk of lifestyle-related diseases. However, the pathogenesis and medication of atherosclerosis have not been elucidated. We developed a rat model of lifestyle-related diseases by feeding a high-fat diet and 30% sucrose solution (HFDS) to spontaneously hypertensive hyperlipidemic rats (SHHR) and reported that this model is a useful model of early atherosclerosis. In order to elucidate the pathogenesis of early atherosclerosis, we searched for atherosclerosis-related genes by microarray analysis using the aortic arch rat model of lifestyle-related diseases. Main methodsFour-month-old male Sprague–Dawley rats and SHHR were each divided into two normal diet (ND) groups and two HFDS groups. After a four-month treatment, the expression of mRNA in the aortic arch was detected using the oligo DNA microarray one-color method and quantified using real-time PCR. Key findingsIn this study, we detected 39 genes in microarray analysis. Esm1, Retnlb Mkks, and Grem2 showed particularly marked changes in gene expression in the SHHR-HFDS group. Compared with the SD-ND group, the SHHR-HFDS group had an increase in Mkks gene expression of about 26-fold and an approximately 22-fold increase in the expression of Grem2. Similarly, the expression of Esm1 increased by about 12-fold and that of Retnlg by about 10-fold as shown by quantitative real-time PCR. SignificanceThis study suggested that these four genes might be important in early atherosclerosis development.

Gene expression of the marine copepod Calanus finmarchicus: Responses to small-scale environmental variation in the Gulf of Maine (NW Atlantic Ocean)

The pelagic copepod Calanus finmarchicus is one of the most important zooplankton species in the North Atlantic Ocean. Despite its ecological importance and pivotal role in the food chain, the molecular mechanisms underlying this species' complex life history (ontogenetic development, reproduction, molting, and diapause) and physiology (digestion, neural processes, and membrane physiology) have remained poorly characterized. This study examined differential expression of nearly 1000 genes, selected based on physiological function and hypothesized ecological importance, for C. finmarchicus collected in the Gulf of Maine (NW Atlantic Ocean) using an oligonucleotide DNA microarray. Replicate analyses compared adult females and final-stage juveniles (Copepodite-V stages) collected from surface (0–30m) and deep (130–170m) layers during April, 2008. Differentially expressed genes were identified by statistical analysis of multiple replicates, including a control for False Discovery Rate (FDR). Functional relationships of selected genes and/or proteins were evaluated using the Gene Ontology Enrichment Analysis Software Toolkit (GOEAST). Genes involved in protein synthesis, cell-cycle and tissue buildup were shown to be up-regulated (i.e., significantly higher expression levels) in deep females and juveniles; genes related to protein turnover, cellular homeostasis, activity, and stress/immunity responses were up-regulated in surface females. Additional functional analyses using KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis indicated up-regulation of genes encoding enzymes related to energy metabolism and osmoregulation in surface females, and citric acid cycle and anaerobic metabolism in deep females and juveniles. Overall, an active physiological state was pronounced in surface females, and a number of processes related with emergence from diapause were pronounced in deep females and juveniles.