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PubMed2004

Quantitative expression analysis of GH3, a gene induced by plant growth regulator herbicides in soybean.

Symptoms resembling off-target plant growth regulator (PGR) herbicide injury are frequently found in soybean fields, but the causal agent is often difficult to identify. The expression of GH3, an auxin-regulated soybean gene, was quantified from soybean leaves injured by PGR herbicides using real-time RT-PCR. Expression of GH3 was analyzed to ascertain its suitability for use in a diagnostic assay to determine whether PGR herbicides are the cause of injury. GH3 was highly induced by dicamba within 3 days after treatment (DAT) and remained high at 7 DAT, but induction was much lower at 17 DAT. GH3 was also highly induced at 7 DAT by dicamba + diflufenzopyr, and to a lesser extent by the other PGR herbicides clopyralid and 2,4-D. The non-PGR herbicides glyphosate, imazethapyr, and fomesafen did not significantly induce GH3 expression above a low constitutive level. These results indicate that a diagnostic assay for PGR herbicide injury based on overexpression of auxin-responsive genes is feasible, and that GH3 is a potential candidate from which a diagnostic assay could be developed. However, time course analysis of GH3 expression indicates the assay would be effective for a limited time after exposure to the herbicide.

Gene Expression↗

PubMed2002

The human sideroflexin 5 (SFXN5) gene: sequence, expression analysis and exclusion as a candidate for PARK3.

Parkinson's disease (PD) is a common neurodegenerative disorder with clinical features of bradykinesia, rigidity and resting tremor resulting from the deficiency of dopamine in the nigrostriatal system. Previously we mapped a susceptibility gene for an autosomal dominant form of PD to a 10.6 cM region of chromosome 2p (PARK3; OMIM 602404). Here we report the identification and characterization of the human sideroflexin 5 gene (SFXN5), which maps to the critical PARK3 region. Database analysis and 5'-RACE (rapid amplification of cDNA ends) identified a 4191 bp cDNA, encoding a predicted protein of 340 amino acids. The genomic sequence and structure of SFXN5 confirmed the cDNA sequence. Northern blot analysis revealed a single SFXN5 transcript of approximately 4.3 kb, which was primarily expressed in the brain. An examination of SFXN5 expression in specific regions of the human brain revealed high levels of expression in all regions analyzed. Sequence analysis of 2p13 linked individuals affected with PD did not reveal any potentially pathogenic mutations within SFXN5, suggesting SFXN5 does not correspond to PARK3.

Amino Acid Sequence↗

PubMed2003

Molecular cloning, transcriptional, and expression analysis of the first cellulase gene (cbh2), encoding cellobiohydrolase II, from the moderately thermophilic fungus Talaromyces emersonii and structure prediction of the gene product.

A gene (cbh2) encoding cellobiohydrolase II was isolated from the fungus Talaromyces emersonii by rapid amplification of cDNA ends techniques and the equivalent genomic sequence was subsequently cloned. This represents the first report of a key component of the cellulase regulon from this organism. DNA sequencing revealed that cbh2 has an open reading frame of 1377 bp, which encodes a putative polypeptide of 459 amino acids, and is interrupted by seven introns. The deduced amino acid sequence revealed that cbh2 has a modular structure with a predicted molecular mass of 47 kDa and consisting of a fungal type carbohydrate binding module separated from a catalytic domain by a proline/serine/threonine rich linker region. The deduced protein is homologous to fungal cellobiohydrolases in Family 6A of the glycosyl hydrolases. Profiles of cbh2 expression in T. emersonii investigated by Northern blot analysis revealed that expression is regulated at the transcriptional level. Expression of the T. emersonii cbh2 gene is induced by cellulose, xylan, xylose, and gentiobiose and clearly repressed by glucose. Putative regulatory element consensus sequences have been identified in the upstream regulatory sequence of the cbh2 gene including the catabolite repressor element and the activator of cellulase expression (Ace) binding sites. High sequence identity (67%) between the catalytic domain of Cel 6A from Trichoderma reesei and the T. emersonii cbh2 gene product allowed structure prediction for the 3D model of the T. emersonii catalytic domain to be a variant of the classical TIM alpha/beta fold.

Amino Acid Sequence↗

PubMed2006

Isolation and expression analysis of a GDSL-like lipase gene from Brassica napus L.

As lipolytic enzymes, GDSL lipases play an important role in plant growth and development. In order to identify their functions and roles, the full-length cDNA of a GDSL lipase gene, designated BnLIP2, was isolated from Brassica napus L. BnLIP2 was 1,300 bp long, with 1,122 bp open reading frame (ORF) encoding 373 amino acid residues. Sequence analysis indicated that BnLIP2 belonged to GDSL family. Southern blot analysis indicated that BnLIP2 belonged to a small gene family in rapeseed genome. RT-PCR analysis revealed that BnLIP2 was a tissue-specific expressing gene during reproductive growth and strongly expressed during seed germination. BnLIP2 expression could not be detected until three days after germination, and it subsequently became stronger. The transcript of this gene was deficient in root of seedlings growing at different stages. When juvenile seedlings were treated by methyl jasmonate (MeJ), salicylic acid (SA) and naphthalene acetic acid (NAA), BnLIP2 expression could not be induced in root. Our study implicates that BnLIP2 probably plays an important role in rapeseed germination, morphogenesis, flowering, but independent of root growth and development.

Amino Acid Sequence↗

PubMed2005

A generic approach for the design of whole-genome oligoarrays, validated for genomotyping, deletion mapping and gene expression analysis on Staphylococcus aureus.

BACKGROUND: DNA microarray technology is widely used to determine the expression levels of thousands of genes in a single experiment, for a broad range of organisms. Optimal design of immobilized nucleic acids has a direct impact on the reliability of microarray results. However, despite small genome size and complexity, prokaryotic organisms are not frequently studied to validate selected bioinformatics approaches. Relying on parameters shown to affect the hybridization of nucleic acids, we designed freely available software and validated experimentally its performance on the bacterial pathogen Staphylococcus aureus. RESULTS: We describe an efficient procedure for selecting 40-60 mer oligonucleotide probes combining optimal thermodynamic properties with high target specificity, suitable for genomic studies of microbial species. The algorithm for filtering probes from extensive oligonucleotides libraries fitting standard thermodynamic criteria includes positional information of predicted target-probe binding regions. This algorithm efficiently selected probes recognizing homologous gene targets across three different sequenced genomes of Staphylococcus aureus. BLAST analysis of the final selection of 5,427 probes yielded >97%, 93%, and 81% of Staphylococcus aureus genome coverage in strains N315, Mu50, and COL, respectively. A manufactured oligoarray including a subset of control Escherichia coli probes was validated for applications in the fields of comparative genomics and molecular epidemiology, mapping of deletion mutations and transcription profiling. CONCLUSION: This generic chip-design process merging sequence information from several related genomes improves genome coverage even in conserved regions.

Algorithms↗

PubMed1997

Expression analysis of mortalin, a unique member of the Hsp70 family of proteins, in rat tissues.

We have investigated the expression of mortalin in rat tissues by Northern analysis, RNA in situ hybridization, and immunohistochemical studies. By Northern assay, the highest level of expression was detected in brain, heart, and skeletal muscle followed by lung, liver, and kidney, and the least level of expression was detected in testis and spleen. RNA in situ and immunohistochemical studies showed that mortalin expression is significantly higher in upper nondividing layers than in the lower basal layers of skin, in neurons and nerve fibers than in surrounding glial cells in brain, in cardiomyocytes than in nonmyocytes in heart, and in interstitial secretory tissue than in germinating follicles in ovary. Such tissue- and cell-specific expression patterns of mortalin coordinates with its earlier reported antiproliferative function in normal cells. However, a deregulation of the expression is observed in rat brain tumor along with the detection of nonpancytosolic mortalin in rat glioma cell line C6. The study suggests that mortalin is involved in pathways that regulate division capacities of cells in vivo.

Animals↗

PubMed2004

Phenotypic and microarray gene expression analysis of tri-dimensional raft-modeled human head and neck squamous cell carcinoma.

OBJECTIVES: To describe the phenotypic and gene expression differences in monolayer and tri-dimensional cultures systems. METHODS: Normal oral epithelial cells (NOEC), primary head and neck squamous cell carcinoma (HNSCC), and HNSCC cell lines were used to create and study modeled tri-dimensional tissue. Using cDNA microarray analysis, monolayer and raft-modeled tri-dimensional HNSCC cell lines were compared. RESULTS: NOEC, HNSCC, and both together can be modeled with tri-dimensional differentiation and cytokeratin characteristics analogous to in vivo tissue. Modeling of primary HNSCC resulted in a morphology of invasive HNSCC with areas of direct collagen invasion and MMP2 expression. Gene array analysis suggests that the individual cell lines themselves are the primary gene expression predictor and not the presence of tri-dimensional tissue architecture. CONCLUSIONS: This tissue culture modeling system approximates the differentiation and tri-dimensional structure of in vivo tissues, and that raft modeled tri-dimensional HNSCC does not have a significantly different gene expression profile than the corresponding monolayer culture.

Carcinoma, Squamous Cell↗

PubMed2006

Gene expression analysis identifies novel genes participating in early murine liver development and adult liver regeneration.

Adult liver tissue regeneration may recapitulate molecular events of liver organogenesis. As gaps in our understanding of the fundamental processes that govern development and regeneration of the liver still exist, we studied gene expression in the developing liver at embryonic day 9.5 post coitum (E d9.5 p.c.). Microarray data from E d9.5 p.c. as well as previously published data from embryonic day 11.5 post coitum (E d11.5 p.c.) and embryonic day 13.5 post coitum (E d13.5 p.c.) were subjected to cluster analysis. This led to the identification of 130 genes which were characterized by continuous expression at all stages of liver development with peak expression of 44 genes at E d9.5 p.c. Five of these genes, previously not known to be associated with early liver development or with adult liver regeneration were selected for further analysis. The expression of the genes was studied by real-time polymerase chain reaction at 0, 2, 4, 6, 12, 24 and 48 hr after partial hepatectomy in the adult liver. Two of the genes, growth arrest protein 43 (GAP43) and paired-like homeodomain transcription factor 2 (Pitx2) were exclusively detected at 24 hr, whereas the genes Twist1, Midkine, and zinc finger protein of cerebellum 1 (Zic1) each showed a specific expression profile in the regenerating liver with peak expressions at 4, 24, and 6 hr, respectively. In summary, we were able to identify novel genes, that may act as regulators during liver formation as well as in the regeneration phase of adult liver. This information may contribute to the development of new targets for the treatment of liver diseases in the future.

Animals↗

PubMed2006

Genome-wide expression analysis detects eight genes with robust alterations specific to bipolar I disorder: relevance to neuronal network perturbation.

The limited number of genome-wide transcriptome analyses using the postmortem brains of bipolar disorder sufferers has not produced a clear consensus on the molecular pathways affected by the disorder. To expand the knowledge in this area, we examined the expression levels of more than 12 000 genes in Brodmann's Area (BA), 46 (dorsolateral prefrontal cortex) from bipolar I disorder and control samples using Affymetrix GeneChips. This analysis detected 108 differentially expressed genes in bipolar brains. Validation studies using quantitative RT-PCR on the two original diagnostic cohorts plus tissue from schizophrenic subjects, confirmed the differential expressions of eight genes (RAP1GA1, SST, HLA-DRA, KATNB1, PURA, NDUFV2, STAR and PAFAH1B3) in a bipolar-specific manner and one gene (CCL3) which was downregulated in both bipolar and schizophrenic brains. Of these, protein levels of RAP1GA1 (RAP1 GTPase activating protein 1) showed a trend of increase in BA46 from bipolar brains, in keeping with mRNA transcript levels. Transmission disequilibrium analysis of the nine genes using 43 single nucleotide polymorphisms (SNPs) in 229 National Institute of Mental Health bipolar trios exposed nominal SNP association and modest empirical haplotypic association (P=0.033) between SST (somatostatin) and disease. Finally, gene network analysis using the currently obtained expression data highlighted cellular growth and nervous system development pathways as potential targets in the molecular pathophysiology of bipolar disorder.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed2005

Bovine hepatocyte growth factor and its receptor c-Met: cDNA cloning and expression analysis in the mammary gland.

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic cytokine that plays a crucial role in the embryonic and postnatal development of various organs including the mammary gland. We cloned bovine HGF and its c-Met receptor cDNAs, and examined their expression during mammary gland development in dairy cows. The 2.5-kbp HGF cDNA clone contained a 2190 bp open reading frame coding a 730 amino acid protein, while the 4.8-kbp c-Met cDNA clone contained a 4152 bp open reading frame coding a 1384 amino acid protein. The bovine HGF and c-Met sequences exhibited more than 87% identity with those of other mammals. RT-PCR analysis revealed ubiquitous expression of both HGF and c-Met mRNAs in various bovine tissues tested. HGF mRNA was detected only in the inactive stage of bovine mammary gland development and not in the developing, lactating, and involuting stages, while c-Met mRNA was detected in the inactive and involuting stages. Immunohistochemical analysis demonstrated that the c-Met protein was found on mammary epithelial cells in the inactive, developing, and involuting stages, and on myoepithelial cells in all stages. These results suggest pivotal roles of HGF and c-Met in the development of bovine mammary gland.

Animals↗

PubMed2001

Single-tube gene-specific expression analysis by high primer density multiplex reverse transcription.

Molecular genetics is rapidly moving from simple identification of a gene of interest to characterization of gene products as components in complex networks. Critical tools for gene product analysis require a rapid method for evaluation of contextual expression. Here, we describe a robust, high primer density, single-tube, multiplex reverse transcription (HD-MRT) technique. This approach is capable of analyzing for the presence of numerous transcripts when polymerase chain reaction (PCR) is subsequently employed for individual gene-specific sequence amplification (HD-MRT-PCR). This assay substantially increases the total number of different cDNAs for amplification beyond previously published techniques. Our approach simultaneously eliminates RNA quality control issues for samples run in parallel while improving efficiency in the use of time and materials. This assay is designed for broad applicability and accessibility, employs modifications of commercially available components, and allows more than 25 independently selected gene-specific primers to be used simultaneously. Our protocol allows multiplexed primers to behave similarly to uniplex RT reactions, while avoiding potential interference between gene-specific and/or nonspecific primers during annealing and reverse transcription. Expression of putatively networked homologous transcripts was analyzed in multiple cell lines and tissues from mouse and human to validate the technique.

Animals↗

PubMed2004

Gene expression analysis of human renal biopsies: recent developments towards molecular diagnosis of kidney disease.

PURPOSE OF REVIEW: The analysis of renal tissue from kidney biopsies by histology, electron microscopy and immunohistology represents the current standards used to establish a specific diagnosis in nephrology. Recent progress in gene expression-based tissue analysis may provide fundamentally novel information in renal biopsy interpretation. In this review, progress towards the routine application of this approach is summarized. RECENT FINDINGS: Renal disease is characterized by closely interrelated mechanisms of inflammation, repair, scarring and atrophy affecting over 20 different intrinsic renal cell types. The renal biopsy sample represents a 'snapshot' of these dynamic processes. A central question for molecular diagnosis is whether specific gene expression patterns can adequately define segments of these disease processes. Can molecular markers be extracted as effectively as has been shown in oncology? Several studies have been able to correlate renal gene expression patterns with clinical parameters, renal histological findings and patient follow-up data. In small populations, molecular markers have been able to provide novel diagnostic, prognostic and differential therapeutic information beyond conventional histology. SUMMARY: A growing number of renal gene expression projects are generating targets for the integration of molecular approaches into kidney biopsy evaluation. If these molecular makers can pass rigorous testing for their diagnostic value, they should become an indispensable part of the management of the renal patient.

Biomarkers↗

PubMed2002

Separation of live cells in different phases of the cell cycle for gene expression analysis.

BACKGROUND: Homogeneity of cell populations is a basic requirement for gene expression analyses of the cell cycle, such as those based on microarrays. The most common approach to obtain specific populations is the use of synchronization methods that increase the number of cells representing a certain cell cycle stage. On the one hand, conventional synchronization usually causes undesirable effects. On the other hand, cell separation methods may imply loss of RNA quality, another limiting factor for expression profiling. We describe a new strategy to specifically separate live cells in different phases of the cell cycle (G(1) and G(2)/M) to obtain good quality RNA for gene expression analyses. METHODS: The experimental design included sorting G(1) and G(2)/M cells with the vital fluorochrome Hoechst 33342, followed by RNA isolation from the sorted cells. RESULTS: Sorted living G(1) and G(2)/M cells, analyzed by immunocytochemistry and laser scanning cytometry, showed strong enrichment. The quality and specificity of the isolated RNA were demonstrated by northern blot. CONCLUSIONS: This new approach has many potential applications, such as expression profiling of specific cell populations after eliminating the irrelevant data produced by cells in other stages of the cycle.

Benzimidazoles↗

PubMed2005

Gene expression analysis of six GC-rich Gram-negative phytopathogens.

Predicted highly expressed (PHX) genes are comparatively analyzed for six GC-rich Gram-negative phytopathogens, i.e., Ralstonia solanacearum, Agrobacterium tumefaciens, Xanthomonas campestris pv. campestris (Xcc), Xanthomonas axonopodis pv. citri (Xac), Pseudomonas syringae pv. tomato, and Xylella fastidiosa. Enzymes involved in energy metabolism, such as ATP synthase, and genes involved in TCA cycle, are PHX in most bacteria except X. fastidiosa, which prefers an anaerobic environment. Most pathogenicity-related factors, including flagellar proteins and some outer membrane proteins, are PHX, except that flagellar proteins are missing in X. fastidiosa which is spread by insects and does not need to move during invasion. Although type III secretion system apparatus are homologous to flagellar proteins, none of them is PHX, which support the viewpoint that the two types of genes have evolved independently. Furthermore, it is revealed that some biosynthesis-related enzymes are highly expressed in certain bacteria. The PHX genes may provide potential drug targets for the design of new bactericide.

Bacterial Proteins↗

PubMed2004

Quantitative gene expression analysis in a nonhuman primate model of antibiotic-induced nephrotoxicity.

Gene expression patterns using microarrays have been described for rodent models of nephrotoxicity. To determine if significant gene expression changes previously identified have application across multiple species, we studied quantitative gene expression changes in the kidneys of female cynomolgus monkeys after exposure to two nephrotoxicants. Animals were dosed with the aminoglycoside gentamicin (10 mg/kg), the experimental oligosaccharide antibiotic everninomicin (30 or 60 mg/kg), or a combination of gentamicin (10 mg/kg) and everninomicin (30 mg/kg) for 7 days. Monkeys receiving these drugs in combination developed renal lesions as early as Day 1. By Day 7, monkeys dosed with 60 mg/kg everninomicin alone also developed renal lesions, while the group exposed to both compounds had more extensive renal damage. The modulation of several genes previously reported to be associated with nephrotoxicity in rodent models was confirmed using quantitative real-time PCR. Among these, waf-1, matrix metalloproteinase-9, and vimentin exhibited changes consistent with the definition of a genomic indicator of toxicity. In addition, we identified three early gene biomarkers that may be predictive of drug-induced nephrotoxicity: clusterin, osteopontin, and hepatitis A virus cellular receptor-1. Logistic regression demonstrated a high degree of correlation between changes in gene expression and the probability of the development of histopathologic lesions. These results are the first confirming rodent gene expression changes associated with nephrotoxicity in a nonhuman primate model and provide preliminary evidence for identifying early gene expression changes predicting the onset of drug-induced renal tubular damage in cynomolgus monkeys.

Aminoglycosides↗

PubMed2002

Facial Expressivity at 4 Months: A Context by Expression Analysis.

The specificity predicted by differential emotions theory (DET) for early facial expressions in response to 5 different eliciting situations was studied in a sample of 4-month-old infants (n = 150). Infants were videotaped during tickle, sour taste, jack-in-the-box, arm restraint, and masked-stranger situations and their expressions were coded second by second. Infants showed a variety of facial expressions in each situation; however, more infants exhibited positive (joy and surprise) than negative expressions (anger, disgust, fear, and sadness) across all situations except sour taste. Consistent with DET-predicted specificity, joy expressions were the most common in response to tickling, and were less common in response to other situations. Surprise expressions were the most common in response to the jack-in-the-box, as predicted, but also were the most common in response to the arm restraint and masked-stranger situations, indicating a lack of specificity. No evidence of predicted specificity was found for anger, disgust, fear, and sadness expressions. Evidence of individual differences in expressivity within situations, as well as stability in the pattern across situations, underscores the need to examine both child and contextual factors in studying emotional development. The results provide little support for the DET postulate of situational specificity and suggest that a synthesis of differential emotions and dynamic systems theories of emotional expression should be considered.

Journal Article↗

PubMed2006

Identification and expression analysis of cold-regulated genes from the cold-hardy Citrus relative Poncirus trifoliata (L.) Raf.

Citrus is a cold-sensitive genus and most commercially important varieties of citrus are susceptible to freezes. On the other hand, Poncirus trifoliata (L.) Raf. is an interfertile Citrus relative that can tolerate temperatures as low as -26 degrees C when fully cold acclimated. Therefore, it has been used for improving cold tolerance in cold-sensitive commercial citrus rootstock varieties and in attempts to improve scion varieties. In this study, cDNA libraries were constructed from both 2-day cold-acclimated and from non-acclimated Poncirus seedlings using a subtractive hybridization method with the objective of identifying cold-regulated genes. A total of 192 randomly picked clones, 136 from the cold-induced library and 56 from the cold-repressed library, were sequenced. The majority of these clones showed sequence homology to previously identified cold-induced and/or environmental stress-regulated genes in Arabidopsis. In addition, some of them shared homology with cold and/or environmental stress-induced genes previously identified in other herbaceous and woody perennial plants and some showed no homology with sequences in GenBank. When these 192 cDNAs were analyzed by reverse northern blot with cold-acclimated and non-acclimated probes, 92 of the cDNAs displayed significantly increased expression, ranging from 2 to 49-fold, during cold acclimation; all 92 were from the cold-induced library. Surprisingly no clones displayed significantly repressed expression in response to cold. Analysis of a number of selected genes individually in northern blots of mRNA from cold-acclimated and non-acclimated plants largely confirmed the reverse northern analysis, verifying induction of expression of selected cDNAs in response to cold. The results showed that subtractive hybridization is an efficient method for identification of cold-induced genes in plants with limited sequence information available. This study also revealed that genes induced during cold acclimation of the cold-hardy citrus relative P. trifoliata are similar to those in Arabidopsis, indicating that similar pathways may be present and activated during cold acclimation in woody perennial plants.

Acclimatization↗

PubMed2004

Gene expression analysis of the lung following paraquat administration in rats using DNA microarray.

Gene expression changes in the lungs induced by paraquat (PQ) administration were studied in rats using DNA microarrays that were detectable for 1,090 genes per DNA microarray. The rats were subjected to subacute PQ exposure (7 mg/kg, s.c., daily for eight administrations). Two days after the final administration, the rats were divided into two groups. Group 1 experienced significant body weight loss and displayed signs of subacute PQ toxicity, but Group 2 showed no significant effects due to the PQ treatment. A control group, Group 3, was also included. In the comparison of the gene expression levels in the animals from Group 1 or Group 2 to the control animals treated by vehicle, 48 genes in Group 1 and 29 genes from Group 2 were differentially expressed. The twenty-eight genes were common to these two groups. These differentially expressed genes following paraquat treatment were classified as follows: 5 neurotransmitter receptor genes; 4 transporter genes; 4 voltage-gated ion channel genes; 2 lipid metabolism enzyme genes; 2 G-proteins involved in endocytosis and exocytosis genes; 7 cytokine genes; 4 ADP ribosylation genes involved in cell death and regeneration; CFTR gene, which is the causal gene for cystic fibrosis; neurofibromatosis type 1 gene, which is the causal gene for the neurofibromatosis type 1 that is known to accompany pulmonary fibrosis; and the causal gene for spinocerebellar ataxia. These genes may prove to be the keys for the elucidation of the mechanism of PQ toxicity, e.g. PQ-induced pulmonary fibrosis.

Animals↗