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PubMed2003

Gene expression analysis in interstitial lung edema induced by saline infusion.

To investigate the molecular events taking place during the development of hydraulic interstitial edema, we analyzed by microarray and conventional molecular techniques the variation of gene expression in lung from rabbits treated with slow-rate saline infusions. This analysis indicates that even a condition characterized by a small increase in extravascular water can have a significant influence on the inflammatory milieu. In this regard, cytokines, in particular TNFalpha, can be considered early mediators capable of inducing secondary effects on the injured tissue. Moreover, two MT1 genes were strongly up-regulated, data consistent with their role as protective molecules.

Animals↗

PubMed2005

Prospective gene expression analysis accurately subtypes acute leukaemia in children and establishes a commonality between hyperdiploidy and t(12;21) in acute lymphoblastic leukaemia.

We have prospectively analysed and correlated the gene expression profiles of children presenting with acute leukaemia to the Royal London and Great Ormond Street Hospitals with morphological diagnosis, immunophenotype and karyotype. Total RNA extracted from freshly sorted blast cells was obtained from 84 lymphoblastic [acute lymphoblastic leukaemia (ALL)], 20 myeloid [acute myeloid leukaemia (AML)] and three unclassified acute leukaemias and hybridised to the high density Affymetrix U133A oligonucleotide array. Analysis of variance and significance analysis of microarrays was used to identify discriminatory genes. A novel 50-gene set accurately identified all patients with ALL and AML and predicted for a diagnosis of AML in three patients with unclassified acute leukaemia. A unique gene set was derived for each of eight subtypes of acute leukaemia within our data set. A common profile for children with ALL with an ETV6-RUNX1 fusion, amplification or deletion of ETV6, amplification of RUNX1 or hyperdiploidy with an additional chromosome 21 was identified. This suggests that these rearrangements share a commonality in biological pathways that maintains the leukaemic state. The gene TERF2 was most highly expressed in this group of patients. Our analyses demonstrate that not only is microarray analysis the single most effective tool for the diagnosis of acute leukaemias of childhood but it has the ability to identify unique biological pathways. To further evaluate its prognostic value it needs to be incorporated into the routine diagnostic analysis for large-scale clinical trials in childhood acute leukaemias.

Acute Disease↗

PubMed2004

Gene expression analysis in the hippocampal formation of tree shrews chronically treated with cortisol.

Adrenal corticosteroids influence the function of the hippocampus, the brain structure in which the highest expression of glucocorticoid receptors is found. Chronic high levels of cortisol elicited by stress or through exogenous administration can cause irreversible damage and cognitive deficits. In this study, we searched for genes expressed in the hippocampal formation after chronic cortisol treatment in male tree shrews. Animals were treated orally with cortisol for 28 days. At the end of the experiments, we generated two subtractive hippocampal hybridization libraries from which we sequenced 2,246 expressed sequenced tags (ESTs) potentially regulated by cortisol. To validate this approach further, we selected some of the candidate clones to measure mRNA expression levels in hippocampus using real-time PCR. We found that 66% of the sequences tested (10 of 15) were differentially represented between cortisol-treated and control animals. The complete set of clones was subjected to a bioinformatic analysis, which allowed classification of the ESTs into four different main categories: 1) known proteins or genes (approximately 28%), 2) ESTs previously published in the database (approximately 16%), 3) novel ESTs matching only the reference human or mouse genome (approximately 5%), and 4) sequences that do not match any public database (50%). Interestingly, the last category was the most abundant. Hybridization assays revealed that several of these clones are indeed expressed in hippocampal tissue from tree shrew, human, and/or rat. Therefore, we discovered an extensive inventory of new molecular targets in the hippocampus that serves as a reference for hippocampal transcriptional responses under various conditions. Finally, a detailed analysis of the genomic localization in human and mouse genomes revealed a survey of putative novel splicing variants for several genes of the nervous system.

Animals↗

PubMed2006

Microarray expression analysis of meiosis and microsporogenesis in hexaploid bread wheat.

BACKGROUND: Our understanding of the mechanisms that govern the cellular process of meiosis is limited in higher plants with polyploid genomes. Bread wheat is an allohexaploid that behaves as a diploid during meiosis. Chromosome pairing is restricted to homologous chromosomes despite the presence of homoeologues in the nucleus. The importance of wheat as a crop and the extensive use of wild wheat relatives in breeding programs has prompted many years of cytogenetic and genetic research to develop an understanding of the control of chromosome pairing and recombination. The rapid advance of biochemical and molecular information on meiosis in model organisms such as yeast provides new opportunities to investigate the molecular basis of chromosome pairing control in wheat. However, building the link between the model and wheat requires points of data contact. RESULTS: We report here a large-scale transcriptomics study using the Affymetrix wheat GeneChip(R) aimed at providing this link between wheat and model systems and at identifying early meiotic genes. Analysis of the microarray data identified 1,350 transcripts temporally-regulated during the early stages of meiosis. Expression profiles with annotated transcript functions including chromatin condensation, synaptonemal complex formation, recombination and fertility were identified. From the 1,350 transcripts, 30 displayed at least an eight-fold expression change between and including pre-meiosis and telophase II, with more than 50% of these having no similarities to known sequences in NCBI and TIGR databases. CONCLUSION: This resource is now available to support research into the molecular basis of pairing and recombination control in the complex polyploid, wheat.

Chromosome Pairing↗

PubMed2006

Identification and expression analysis of rainbow trout pumilio-1 and pumilio-2.

Pumilio is a sequence-specific RNA-binding protein that regulates translation from the relevant mRNA. The PUF-domain, the RNA-binding motif of Pumilio, is highly conserved across species. In the present study, we have identified two pumilio genes (pumilio-1 and pumilio-2) in rainbow trout and analyzed their expression patterns in its tissues. Pumilio-1 mRNA and pumilio-2A mRNA code for typical full length Pumilio proteins that contain a PUF-domain, whereas pumilio-2B mRNA is a splice variant of pumilio-2 and encodes a protein that lacks the PUF-domain. We have also identified a novel 72-bp exon that has not been reported in other animal species but is conserved in fish species. The insertion of this novel exon leads to the expression of an isoform of the Pumilio-2 protein with a slightly altered conformation of the PUF-domain. Pumilio-1 mRNA and pumilio-2A mRNA (irrespective of the presence of the 72-bp exon) are expressed in both the brain and ovaries at high levels, whereas pumilio-2B mRNA is expressed at low levels in all the rainbow trout tissues examined. Western blot analysis also indicates that the full length Pumilio proteins are expressed predominantly in the brain and ovaries. These data suggest that the Pumilio proteins have physiological roles and are involved in regulatory mechanisms in rainbow trout.

Alternative Splicing↗

PubMed2003

[Molecular cloning for testis spermatogenesis cell apoptosis related gene TSARG1 and Mtsarg1 and expression analysis for Mtsarg1 gene].

Spermatogenesis cell apoptosis is a very complex process, which needs many molecules to take part in the programmable death of cells in testis. At present, research of apoptosis for spermatogenesis cell is at the primary step. It is very important to clone spermatogenesis cell apoptosis related genes and spermatogenesis genes in testis. Applying the bioinformatics and experiment technique, we have cloned human and mouse novel gene cDNA sequences--human testis and spermatogenesis cell apoptosis related gene 1 (TSARG1) and mouse testis and spermatogenesis cell apoptosis related gene 1(Mtsarg1) from human and mouse testis cDNA library respectively, using a cDNA fragment (GenBank accession number: BE644538) as an electronic probe, which was significantly changed in expression in cryptorchidism. The GeneBank accession numbers of Mtsarg1 and TSARG1 are AF399971 and AY032925 (NM_139073), respectively. The Mtsarg1 has a 55% identity and 61% similarity with TSARG1 at the amino acid level, which did not share significant homology with any other known protein in databases. The full-length cDNA of TSARG1 gene is 973 bp, including 549 bp open reading frame(ORF) and coding 183 amino acids, whereas the full-length cDNA of Mtsarg1 gene is 1103 bp, including 576 bp ORF and coding 192 amino acids. The predicted molecule weight of TSARG1 is 19948.61 Dolton, and the deduced iso-electric point is 10.24, whereas the Mtsarg1 is 20875.93 and is 9.83, being alkaline proteins. RT-PCR analysis showed that Mtsarg1 was expressed significantly in testis and faintly in epididymis in the ten tissues of testis, ovary, spleen, kidney, lung, heart, brain, epididymis, liver and skeletal muscle in mouse, while it wasn't expressed in the other eight tissues. Therefore, our results suggested that Mtsarg1 and TSARG1 would be pay potential roles in spermatogenesis cell apoptosis or spermatogenesis.

Amino Acid Sequence↗

PubMed2002

Cloning and expression analysis of the chick DAN gene, an antagonist of the BMP family of growth factors.

Differential screening-selected gene aberrative in neuroblastoma (DAN) is a member of a cystine knot protein family that includes Cerberus and Gremlin. First isolated in a screen to identify genes down-regulated in transformed rat fibroblasts, DAN has subsequently been cloned in Xenopus, mouse, and human. Overexpression of DAN suppresses the transformed phenotype and retards the cell's entry into S phase. Biochemical analyses have demonstrated DAN's ability to bind bone morphogenetic proteins and antagonize their signaling activity. In this study, chick DAN was cloned and sequenced, revealing a conserved cystine knot region as well as an N-glycosylation site. A riboprobe was designed from the 3' chick DAN coding sequence and used for analysis of DAN in the developing chick embryo by in situ hybridization. Chick DAN was expressed beginning at stage 10 in the developing somites and the medial otic epithelium. Expression in the neural layer of the eye became apparent at stage 14. By stage 17, expression had expanded to the base of the hindbrain. Limb bud labeling began at stage 20, whereas expression in the branchial arches appeared at stage 25. Chick DAN expression generally corresponded to that of mouse DAN expression as shown by comparative in situ hybridization. However, chick DAN was found in the otic epithelium and notochord, whereas mouse DAN was restricted to the overlying otic ectomesenchyme and was absent from the notochord. This observation suggests that DAN may play different roles in chick and mouse otic and notochord development.

Animals↗

PubMed2002

Sequence and expression analysis of deer mouse interferon-gamma, interleukin-10, tumor necrosis factor, and lymphotoxin-alpha.

Deer mice (Peromyscus maniculatus) are the principal host species of Sin Nombre (SN) virus, the primary etiologic agent of hantavirus cardiopulmonary syndrome in North America. The disease is a cytokine-mediated immunopathology characterized by pulmonary mononuclear infiltrates without discernible viral pathology. Infected deer mice remain life-long carriers and virus is found in many organs, including the lungs, but without pathology. It is unclear how deer mice respond to SN virus because no tools exist to examine the immune response in infected animals. As an initial step in examining host responses to SN virus, we have cloned partial cDNAs of deer mouse interferon-gamma (IFN-gamma), interleukin-10 (IL-10), tumor necrosis factor (TNF) and lymphotoxin-alpha (LTalpha). IL-10, TNF and LTalpha sequences are highly conserved compared to orthologs of other mammalian species, while IFN-gamma is substantially less conserved. Phylogenetic analyses indicate that the amino acid sequences of IFN-gamma and TNF may be useful in resolving relationships at the subfamily level within the rodent family Muridae. While all four sets of analyses were able to reconstruct clade Rodentia, they were not able to resolve the relationships among the mammalian orders represented in this study. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of concanavalin A-stimulated splenocytes determined that maximal IFN-gamma and TNF expression occurred rapidly while IL-10 and LTalpha expression was maximal at 24 h.

Amino Acid Sequence↗

PubMed1995

Expression analysis, genomic structure, and mapping to 7q31 of the human sperm adhesion molecule gene SPAM1.

During the course of systematic sequence tag analysis of clones isolated from an adult testis cDNA library, clones 296 and 576 were found to detect 71-74% sequence identity to the guinea pig sperm surface protein PH-20. This surface protein is involved in sperm-egg adhesion in the guinea pig. Nucleotide sequence for 1919 bp of human DNA from a series of overlapping cDNA clones isolated from a testis cDNA library confirmed the sequence identity within a 1527-bp open reading frame to be 71-74% to the guinea pig gene and the similarity to be 60% for the predicted protein of 509 amino acids. Southern blot analysis of human genomic DNA and DNA from somatic cell hybrids indicates that the gene (SPAM1) is unique and does not form part of a larger family and that it maps to chromosome 7. Fluorescence in situ hybridization with yeast artificial chromosome (YAC) clones isolated from the CEPH megaYAC library has refined this localization to 7q31. PCR analysis of genomic DNA and YAC clone DNA has shown that the 1919 bp of the gene that has been cloned covers approximately 11 kb of genomic DNA and is encoded by at least 4 exons. Northern analysis of poly(A)+ mRNA from a range of 16 human tissues has demonstrated that expression of the gene as a single 2.4-kb transcript is strictly limited to the testis.

Adult↗

PubMed2006

Regenerative growth is impacted by TCDD: gene expression analysis reveals extracellular matrix modulation.

Adult zebrafish can completely regenerate their caudal fin following amputation. This complex process is initiated by the formation of an epithelial wound cap over the amputation site by 12 h post amputation (hpa). Once the cap is formed, mesenchymal cells proliferate and migrate from sites distal to the wound plane and accumulate under the epithelial cap forming the blastemal structure within 48 hpa. Blastemal cells proliferate and differentiate, replacing the amputated tissues, which are populated with angiogenic vessels and innervating nerves during the regenerative outgrowth phase which is completed around 14 days post amputation (dpa). Regenerative outgrowth does not occur in TCDD-exposed zebrafish. To identify the molecular pathways that are perturbed by TCDD exposure, male zebrafish were ip injected with 50 ng/g TCDD or vehicle and caudal fins were amputated. Regenerating fin tissue was collected at 1, 3, and 5 dpa for mRNA abundance analysis. Microarray analysis and quantitative real time PCR revealed that wound healing and regeneration alone altered the expression of nearly 900 genes by at least two-fold between 1 and 5 dpa. TCDD altered the abundance of 370 genes at least two-fold. Among these, several known aryl hydrocarbon responsive genes were identified in addition to several genes involved in extracellular matrix composition and metabolism. The profile of misexpressed genes is suggestive of impaired cellular differentiation and extracellular matrix composition potentially regulated by Sox9b.

Animals↗

PubMed1997

Expression analysis of a gene family in loblolly pine (Pinus taeda L.) induced by water deficit stress.

A cDNA clone (lp3) from loblolly pine induced by water deficit stress (WDS) has been isolated. It is preferentially induced in roots with a constitutive basal level of expression also observed in stems and needles. Northern blot analysis with well irrigated ABA-treated seedlings indicated that the overall accumulation of lp3 transcripts in the roots was lower than that of water deficit-stressed seedlings. However, within roots, lp3 was induced by ABA indicating that the expression of lp3 in roots under WDS conditions was partly mediated by ABA. The lp3 clone is similar to a group of genes called asr (ABA stress and ripening) genes identified in several species. A genomic clone (lp3-1) was identified and its putative protein has the hydrophylicity profile similar to that of lp3 except for two deletions in the 5' region. The genomic Southern and RT-PCR (reverse transcriptase-polymerase chain reaction) analyses indicate that the lp3 gene belongs to a small multigene family of at least four members with a distinct pattern of expression during WDS.

Amino Acid Sequence↗

PubMed2006

Profiles of novel diurnally regulated genes in mouse hypothalamus: expression analysis of the cysteine and histidine-rich domain-containing, zinc-binding protein 1, the fatty acid-binding protein 7 and the GTPase, ras-like family member 11b.

Gene expression profiling of suprachiasmatic nucleus, ventrolateral preoptic area and the lateral hypothalamus was used to identify genes regulated diurnally in the hypothalamus of Mus musculus. The putative transcription regulator, cysteine and histidine-rich domain-containing, zinc binding protein 1, which had not been previously described in brain, was found to cycle diurnally in hypothalamus and forebrain with peak levels of mRNA expression during the dark phase. mRNA for the brain-type fatty acid binding protein 7 was found to change rhythmically in hypothalamic and extra-hypothalamic brain regions reaching peak levels early in the light phase suggesting that lipid metabolism is under circadian regulation in astrocytes. Rhythmically expressed genes in suprachiasmatic nucleus identified here were compared with previous reports in a meta-analysis. Genes held in common included fabp7, and the period gene, Per2. Also identified were genes implicated in guanosine-mediated signaling pathways that included dexamethasone-induced ras-related protein one (dexras1), regulator of G-protein signaling (rgs) 16, and ras-like family member 11b. Northern blotting confirmed diurnal changes in mRNA expression in the hypothalamus for these genes. Ras-like family member 11b was examined in more detail using in situ hybridization and antiphase diurnal changes in expression in suprachiasmatic nucleus and arcuate nucleus were identified implicating the gene in circadian-related, guanosine-mediated signaling. The transcription transactivator protein, CBP/p300-interacting transactivators with glutamic acid/aspartic acid-rich carboxyl-terminal domain, which had not been previously identified in brain, was enriched in suprachiasmatic nucleus and discrete regions of the hypothalamus and forebrain. The potential regulatory role of CBP/p300-interacting transactivators with glutamic acid/aspartic acid-rich carboxyl-terminal domain in the transcription of genes like TGF-alpha implicates the protein in diurnal activity rhythms. These results demonstrate the ability of gene expression profiling to identify potential candidates important in circadian or homeostatic processes.

Animals↗

PubMed2001

Biochemical characterization and expression analysis of the Xenopus laevis corticotropin-releasing hormone binding protein.

Corticotropin-releasing hormone (CRH) plays a key role in the regulation of responses to stress. The presence of a high affinity binding protein for CRH (CRH-BP) has been reported in mammals. We have characterized the biochemical properties and expression of CRH-BP in the South African clawed frog, Xenopus laevis. Apparent inhibition constants (K(i[app])) for different ligands were determined by competitive binding assay. Xenopus CRH-BP (xCRH-BP) exhibited a high affinity for xCRH (K(i[app])=1.08 nM) and sauvagine (1.36 nM). Similar to rodent and human CRH-BPs, the frog protein binds urotensin I and urocortin with high affinity, and ovine CRH with low affinity. RT-PCR analysis showed that xCRH-BP is expressed in brain, pituitary, liver, tail, and intestine. Brain xCRH-BP mRNA is expressed at a relatively constant level throughout metamorphosis and increases slightly in the metamorphic frog. By contrast, the gene is strongly upregulated in the tail at metamorphic climax. Thus, regulation of xCRH-BP gene expression is tissue specific. Because xCRH-BP binds CRH-like peptides with high affinity the protein may regulated, the bioavailability of CRH in amphibia as it does in mammals.

Amino Acid Sequence↗

PubMed1993

Regulation of TL antigen expression. Analysis of the T18d promoter region and responses to IFN-gamma.

Type I (alpha/beta) and type II (gamma) IFN enhance MHC class I gene expression through an IFN-responsive element (IRE) present in the 5' flanking region of the class I-a genes. Comparison of the 5' sequences between classical class I-a genes and T region class I-b genes reveals little homology except for presence of a potential IRE. We have found that cell surface expression of thymus leukemia Ag (TL) was up-regulated by IFN-gamma to a greater extent than H-2K,D in all TL+ T cell lines tested. In contrast, IFN-alpha/beta, which significantly increased H-2K and H-2D Ag expression, had only minor effects on TL expression. Resting peripheral T cells, which were considered to be TL- from previous studies, were found to express TL at a low level as determined by flow cytometry, immunoprecipitation, as well as polymerase chain reaction; the level of expression also could be elevated by IFN-gamma. To examine the control of TL gene transcription and its regulation by IFN-gamma, varying lengths of the T18d 5' flanking region were analyzed in chloramphenicol acetyl transferase assays. By deletion analysis, promoter activity and IFN-gamma responsiveness were localized to an 86-bp fragment that contains the IRE. Both responses were localized further to a 32-bp fragment that contained the IRE at its 3' end. RNase protection assays revealed two major transcription initiation sites, one immediately 5' of the IRE and another approximately 60 bp downstream. Furthermore, polymerase chain reaction analysis of mRNA from resting T cells, thymocytes, and T cell tumor lines confirmed the RNase protection data. Thus, transcription of T18d initiates much further upstream than the classical class I genes, can utilize an unusual promoter element, and can be elevated by IFN-gamma.

Animals↗

PubMed2004

Isolation, characterization and expression analysis of a leaf-specific phosphoenolpyruvate carboxylase gene in Oryza sativa.

Suppression subtractive hybridization was carried out to enrich gene fragments over-expressed in rice leaves by subtraction to rice roots, from which two identical cDNA fragments were identified to encode putative phosphoenolpyruvate carboxylase. Then the corresponding full-length cDNA (Osppc) is isolated by RT-PCR and sequenced, which indicates an open reading frame of 2895bp is contained. Its deduced protein is encoded in 10 exons and shows high similarity to many other plant PEPCs. Comparing with maize and bacterial PEPCs, it is revealed that OSPPC shares many conserved domains and active sites that responsible for the structure, activity and regulation of this enzyme. Phylogenetic analysis demonstrates that OSPPC is grouped with C3 form PEPCs of wheat, maize and sorghum, which is consistent with the classification of rice. And a putative promoter element is predicted with DOF binding box, CAAT box and TATA box in the 5'-flanking sequence of Osppc gene. Moreover, Quantitative RT-PCR analyses are performed in hybrid rice and its parents, which show that Osppc is specifically expressed in leaf including leaf vein and sheath.

Amino Acid Sequence↗

PubMed2002

Gene expression analysis in microdissected renal tissue. Current challenges and strategies.

The architecture and compartmentalization of the kidney has stimulated the development of an array of microtechniques to study the functional differences between the distinct nephron segments. With the vast amounts of genomic sequence data now available, the groundwork has been laid for a comprehensive characterization of the molecular pathways defining the differences in nephron function. With the development of sensitive gene expression techniques the tools for a comprehensive molecular analysis of specific renal microenvironments have been provided: Quantitative RT-PCR technologies now allow the analysis of specific mRNAs from as little as single microdissected renal cells. A more global view of gene expression regulation is a logical development from the application of large scale profiling techniques. In this review, we will discuss the power and pitfalls of these approaches, including their potential for the functional characterization of nephron heterogeneity and diagnostic application in renal disease.

Dissection↗

PubMed2004

Molecular cloning, genomic structure, and expression analysis of the mouse transcriptional intermediary factor 1 gamma gene.

Human transcriptional intermediary factor 1 gamma (Tif1gamma), also known as Ret fused gene 7 (RFG7), is a member of a novel family of transcriptional coregulator-encoding genes which function in cell differentiation and development. Here, we report the structure and expression pattern of the mouse Tif1gamma gene. This gene comprises 20 coding exons spanning about 77 kb of genomic DNA on chromosome 3F2, and encodes a 1142-amino-acid protein with 96% identity to the human protein. The locations of exon/intron boundaries correlated well with those for the regions of conserved amino acid sequences (RBCC motif, PHD finger and bromodomain). In situ hybridization analysis of the TIF1gamma mRNA on sections from staged mouse embryos revealed a low level of ubiquitous expression at midgestation, and higher expression levels within the brain and spinal cord epithelium at later developmental stages. Prominent expression was also found in developing sensory epithelia (cochlea, retina, olfactory epithelium), and in several developing organs including the thymus, lung, stomach, intestine, liver, and kidney cortex. In the adult mouse, Tif1gamma mRNA was detected by Northern blot analysis in all tissues examined, with the highest expression level in testis. In situ hybridization and immunohistochemistry studies revealed that expression of the Tif1gamma mRNA and protein varied according to the stage of the seminiferous epithelium cycle. Taken together, these results indicate-and serve as a basis for investigating-a possible involvement of Tif1gamma in the control of embryonic development and spermatogenesis.

Amino Acid Sequence↗

PubMed2003

Genome-wide expression analysis in Corynebacterium glutamicum using DNA microarrays.

DNA microarray technology has become an important research tool for microbiology and biotechnology as it allows for comprehensive DNA and RNA analyses to characterize genetic diversity and gene expression in a genome-wide manner. DNA microarrays have been applied extensively to study the biology of many bacteria including Mycobacterium tuberculosis, but only recently have they been used for the related high-GC Gram-positive Corynebacterium glutamicum, which is widely used for biotechnological amino acid production. Besides the design and generation of microarrays as well as their use in hybridization experiments and subsequent data analysis, recent applications of DNA microarray technology in C. glutamicum including the characterization of ribose-specific gene expression and the valine stress response will be described. Emerging perspectives of functional genomics to enlarge our insight into fundamental biology of C. glutamicum and their impact on applied biotechnology will be discussed.

Corynebacterium↗