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Differential Display (differential + display)
Terms modified by Differential Display Selected AbstractsInflammatory bowel disease-associated gene expression in intestinal epithelial cells by differential cDNA screening and mRNA displayINFLAMMATORY BOWEL DISEASES, Issue 5 2003Kouhei Fukushima M.D., Ph.D. Abstract Intestinal epithelial cells are actively involved in the pathogenesis of inflammatory bowel disease resulting in an altered functional phenotype. The modulation of epithelial gene expression may occur as a consequence of proliferative, metabolic, immune, inflammatory, or genetic abnormalities. Differential screening of epithelial-cell-derived cDNA libraries (from control, ulcerative colitis, and Crohn's disease epithelial cells) and differential display of mRNA were used for investigation of disease-associated gene expression and modulation. Intestinal epithelial gene expression was successfully analyzed by both approaches. Using differential screening with clones encoding mitochondrial genes, quantitative overexpression was observed in both ulcerative colitis and Crohn's disease, while a unique expression of small RNA was noticed in Crohn's disease cells using Alu-homologous clones. Differential display demonstrated that several genes were differentially displayed among control, ulcerative colitis, and Crohn's disease epithelial cells. This was confirmed by immunohistochemical staining of pleckstrin, desmoglein 2 and voltage-dependent anion channel in control and inflammatory bowel disease mucosal samples. In summary, several inflammatory bowel disease-related associations were found. Since both differential screening and display have advantages and limitations, the combination of both techniques can generate complementary information, facilitate search for novel genes, and potentially identify genes uniquely associated with inflammatory bowel disease. [source] Identification and Regulation of Genes from a Biocontrol Strain of Fusarium oxysporumJOURNAL OF PHYTOPATHOLOGY, Issue 9 2007D. R. Fravel Abstract Differential display with three time points revealed that thiram altered expression of numerous genes in the biocontrol fungus Fusarium oxysporum CS-20. Of the 101 bands purified from the differential display gel, 86 were successfully cloned, and 64 sequenced. Based on nucleic acid sequences, homology to known products was found using BLASTn for 26 sequences and homology to hypothetical proteins was found for six sequences, also from Gibberella zeae. One band (BM1 24-1) showed homology to an ABC transporter from three different fungi. Because of its association with detoxification functions, the ABC transporter was selected for further study. Mycelia of CS-20 were exposed to 25 ,g active ingredient (a.i.) thiram in liquid culture for various times from 0 to 8 h. Quantitative real-time PCR was used to evaluate gene expression. At 30 min after treatment with thiram, the ABC transporter was upregulated 20- to 25-fold relative to the control treatment. The ABC transporter was upregulated 15-fold at 1 h after treatment and 10-fold at 2 h. At 8 h after treatment, there was no difference between treated and non-treated for expression of the ABC transporter. Transcription of the gene encoding EST BM1 24-1 is induced in response to thiram treatment and may function in providing resistance in F. oxysporum isolate CS-20 to fungicides and other toxins. Tolerance to toxins may be critical to the successful inclusion of CS-20 in disease control strategies in cropping systems. [source] SREA is involved in regulation of siderophore biosynthesis, utilization and uptake in Aspergillus nidulansMOLECULAR MICROBIOLOGY, Issue 5 2001Harald Oberegger Under conditions of low iron availability, most fungi excrete siderophores in order to mobilize extracellular iron. We show that lack of the GATA-type transcription factor SREA in Aspergillus nidulans not only leads to derepression of siderophore biosynthesis but also to deregulation of siderophore-bound iron uptake and ornithine esterase expression. Furthermore, SREA deficiency causes increased accumulation of ferricrocin, the siderophore responsible for intracellular iron storage. In sreA deletion strains, extracellular siderophore production is derepressed but still regulated negatively by iron availability, indicating the presence of an additional iron-regulatory mechanism. In contrast, iron affects ferricrocin accumulation in a positive way, suggesting a protective role for this siderophore in detoxification of intracellular iron excess. The harmfulness of deregulated iron uptake in this mutant is demonstrated by increased expression of genes encoding the antioxidative enzymes catalase CATB and the superoxide dismutases SODA and SODB. It is noteworthy that iron starvation was found to repress catB expression in wild-type (wt) and SREA-deficient strains, consistent with catB being subject to SREA-independent iron regulation. Differential display led to the identification of putative SREA target genes amcA and mirA. The deduced MIRA amino acid sequence displays significant similarity to recently characterized siderophore permeases of Saccharomyces cerevisiae. amcA encodes a putative mitochondrial carrier for the siderophore precursor ornithine, indicating cross-regulation of siderophore and ornithine metabolism. [source] Cloning of nodule-specific cDNAs of Galega orientalisPHYSIOLOGIA PLANTARUM, Issue 4 2002Seppo Kaijalainen Differential display was applied in order to clone cDNAs expressed exclusively or predominantly in nodules, compared to uninoculated root tissue of Galega orientalis. Forty-five fragments were unique for nodule RNA. These fragments were reamplified and cloned. Six of them produced a nodule-specific signal on Northern hybridization. These six fragments were sequenced. Five of the sequenced fragments showed homology to nodulin-gene sequences in databases, among them Vicia faba mRNA for protein showing partial homology with Medicago sativa nodulin-25 (Nms25), Pisum sativum PsN466, V. faba CCP2 and CCP4, P. sativum ENOD3, and Maackia amurensis ENOD2. The remaining sequence had no significant homology with sequences in the databanks. Full-size cDNA for the homologue to V. faba mRNA for the protein showing partial homology with M. sativa nodulin-25 (Nms25) and P. sativum PsN466 were cloned and sequenced. [source] mRNA Encoding a Putative RNA Helicase of the DEAD-Box Gene Family is Up-Regulated in Trypomastigotes of Trypanosoma cruziTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 6 2000ALBERTO M. DÍAZ AÑEL ABSTRACT. Differential display of mRNAs from Trypanosoma cruzi epimastigote and metacyclic trypomastigote stages showed several mRNA species differing in their expression level. The cDNA corresponding to one of these mRNAs was used as a probe in Northern blots and identified a RNA product of 2.6 kb with an expression level eight or more times higher in trypomastigotes than in epimastigotes. This probe was also used to screen a genomic library of T. cruzi CL Brener clone prepared in lambda FIX. A clone of about 15 kb was selected that, after partial sequencing, revealed an open reading frame of 688 amino acids encoding a deduced protein with similarity to RNA helicases of the DEAD-box gene family. The presence of the eight conserved motifs characteristic of the DEAD protein family was observed in the T. cruzi sequence, indicating that it corresponds to a putative RNA helicase gene, which we named HelTc. Southern blot analysis indicated that HelTc is a single-copy gene. Pulsed-field gel electrophoresis separation of chromosomes of several isolates of T. cruzi showed that this gene was localized in one or two chromosomal bands. [source] Identification of novel markers expressed during fin regeneration by microarray analysis in medaka fishDEVELOPMENTAL DYNAMICS, Issue 9 2007Masanobu Nishidate Abstract Urodeles and fish have a remarkable ability to regenerate lost body parts, whereas many higher vertebrates, including mammals, retain only a limited capacity. It is known that the formation of specialized cell populations such as the wound epidermis or blastema is crucial for regeneration; however, the molecular basis for their formation has not been elucidated. Recently, approaches using differential display and microarray have been done in zebrafish for searching molecules involved in regeneration. Here, we used the medaka fish, a distantly diverged fish species, for microarray screening of transcripts up-regulated during regeneration. By setting criteria for selecting transcripts that are reliably and reproducibly up-regulated during regeneration, we identified 140 transcripts. Of them, localized in situ expression of 12 transcripts of 22 tested was detected either in differentiating cartilage, basal wound epidermis, or blastema. Our results provide useful molecular markers for dissecting the regeneration process at a fine cellular resolution. Developmental Dynamics 236:2685,2693, 2007. © 2007 Wiley-Liss, Inc. [source] An SNF2 factor involved in mammalian development and cellular proliferationDEVELOPMENTAL DYNAMICS, Issue 1 2001Eric H. Raabe Abstract Members of the SNF2 (Sucrose Non-Fermenter) family of chromatin-remodeling proteins function in processes ranging from DNA repair to transcription to methylation. Using differential display, we recently identified a novel member of the SNF2 family that is highly expressed at the mRNA level in proliferating cells and is down-regulated during apoptosis. We have named this gene PASG (Proliferation-Associated SNF2-like Gene). Northern blot analysis of adult mouse tissues shows PASG to be highly expressed in proliferating organs such as thymus, bone marrow, and testis and absent from nonproliferative tissues such as brain and heart. In situ hybridization analysis of mouse embryos shows that PASG is differentially expressed during development, with highest expression in developing face, limbs, skeletal muscle, heart, and tail. In vitro, PASG expression correlates with a shift from a quiescent to a proliferative state. Mice null for PASG (also known as LSH or Hells) are reported to die perinatally, although the mechanism for lethality is unclear (Geiman and Muegge, 2000). To test the hypothesis that PASG functions in cell proliferation, we compared 5-bromodeoxyuridine (BrdU) incorporation in C33A cells transiently transfected with PASG versus empty vector and found that PASG transfected cells showed a significant decrease in the amount of BrdU incorporation. These findings suggest that PASG plays a role in cell proliferation and may function in the development of multiple cell lineages during murine embryogenesis. © 2001 Wiley-Liss, Inc. [source] Selenium affects biosilica formation in the demosponge Suberites domunculaFEBS JOURNAL, Issue 15 2005Effect on gene expression, spicule formation Selenium is a trace element found in freshwater and the marine environment. We show that it plays a major role in spicule formation in the demosponge Suberites domuncula. If added to primmorphs, an in vitro sponge cell culture system, it stimulates the formation of siliceous spicules. Using differential display of transcripts, we demonstrate that, after a 72-h exposure of primmorphs to selenium, two genes are up-regulated; one codes for selenoprotein M and the other for a novel spicule-associated protein. The deduced protein sequence of selenoprotein M (14 kDa) shows characteristic features of metazoan selenoproteins. The spicule-associated protein (26 kDa) comprises six characteristic repeats of 20 amino acids, composed of 10 distinct hydrophobic regions (, 9 amino acids in length). Recombinant proteins were prepared, and antibodies were raised against these two proteins. Both were found to stain the central axial filament, which comprises the silicatein, as well as the surface of the spicules. In the presence of selenium, only the genes for selenoprotein M and spicule-associated protein are up-regulated, whereas the expression of the silicatein gene remains unchanged. Finally we show that, in the presence of selenium, larger silica aggregates are formed. We conclude that selenium has a stimulatory effect on the formation of siliceous spicules in sponges, and it may be involved in the enzymatic synthesis of biosilica components. [source] Differentially expressed genes associated with CIS -diamminedichloroplatinum (II) resistance in head and neck cancer using differential display and CDNA microarrayHEAD & NECK: JOURNAL FOR THE SCIENCES & SPECIALTIES OF THE HEAD AND NECK, Issue 3 2003Eisaku Higuchi MD Abstract Background. The mechanism by which cancer cells become resistant to cis -Diamminedichloroplatinum (II) (cDDP) is not completely understood. To investigate the molecular markers involved in the cDDP resistance, we compared the gene expression profiles between a head and neck squamous cell carcinoma (HNSCC) line sensitive to cDDP and its cDDP-resistant variant. Methods. Both a fluorescent differential display and a cDNA microarray analysis were applied to distinguish the gene profiles between KB, a human HNSCC line, and its cDDP-resistant variant (KB/cDDP). These results were confirmed by Northern blot analysis. Results. One up-regulated gene, glycoprotein hormone ,-subunit, and two down-regulated genes coding membrane proteins, human folate receptor and tumor-associated antigen L6, were identified in KB/cDDP cells. Conclusions. Our findings suggest that development of the cDDP-resistant phenotype is accompanied by alternations of gene expression including a glycoprotein hormone and membrane proteins. These gene products could be new molecular markers for resistance to cDDP. © 2003 Wiley Periodicals, Inc. Head Neck 25: 187,193, 2003 [source] MRNA differential display identification of thyroid hormone-responsive protein (THRP) gene in association with early phase of long-term potentiationHIPPOCAMPUS, Issue 6 2001Y.P. Tang Abstract The process of long-term potentiation (LTP) consists of the early induction and late maintenance phases. Few studies have examined the cellular mechanisms underlying these two phases; their respective mRNA expression profiles have not yet been elucidated. Here we used the technique of PCR differential display to identify genes that are differentially expressed between the early and late phases of LTP in vivo. Our results indicated that the cDNA fragment corresponding to one mRNA with preferentially increased expression during the early, but not late, phase of LTP encodes the rat thyroid hormone-responsive protein (THRP) gene. In situ hybridization analysis confirmed the results obtained from the PCR differential display. Prior NMDA receptor blockade with MK801 prevented induction of LTP and decreased THRP mRNA expression in the dentate gyrus, as assayed by quantitative RT-PCR analysis. THRP antisense oligonucleotide treatment before tetanic stimulation also prevented induction of LTP. However, when THRP antisense oligonucleotide was administered after induction of LTP, it did not affect expression and maintenance of LTP. THRP is known to be responsive to thyroid hormone. Our results indicate that direct thyroid hormone (T3) injection into the dentate gyrus produces a long-lasting enhancement of synaptic efficacy of these neurons. T3 injection also markedly increased THRP mRNA expression in the dentate gyrus. Taken together, our results suggest that THRP mRNA expression plays an important role in the early phase, but not the late phase, of LTP and that both THRP and thyroid hormone are involved in synaptic plasticity in hippocampal neurons. Hippocampus 2001;11:637,646. © 2001 Wiley-Liss, Inc. [source] Inflammatory bowel disease-associated gene expression in intestinal epithelial cells by differential cDNA screening and mRNA displayINFLAMMATORY BOWEL DISEASES, Issue 5 2003Kouhei Fukushima M.D., Ph.D. Abstract Intestinal epithelial cells are actively involved in the pathogenesis of inflammatory bowel disease resulting in an altered functional phenotype. The modulation of epithelial gene expression may occur as a consequence of proliferative, metabolic, immune, inflammatory, or genetic abnormalities. Differential screening of epithelial-cell-derived cDNA libraries (from control, ulcerative colitis, and Crohn's disease epithelial cells) and differential display of mRNA were used for investigation of disease-associated gene expression and modulation. Intestinal epithelial gene expression was successfully analyzed by both approaches. Using differential screening with clones encoding mitochondrial genes, quantitative overexpression was observed in both ulcerative colitis and Crohn's disease, while a unique expression of small RNA was noticed in Crohn's disease cells using Alu-homologous clones. Differential display demonstrated that several genes were differentially displayed among control, ulcerative colitis, and Crohn's disease epithelial cells. This was confirmed by immunohistochemical staining of pleckstrin, desmoglein 2 and voltage-dependent anion channel in control and inflammatory bowel disease mucosal samples. In summary, several inflammatory bowel disease-related associations were found. Since both differential screening and display have advantages and limitations, the combination of both techniques can generate complementary information, facilitate search for novel genes, and potentially identify genes uniquely associated with inflammatory bowel disease. [source] Application of differential display, with in situ hybridization verification, to microscopic samples of breast cancer tissueINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 5 2003Ruey Ho Kao Summary., The technique of differential display (DD) has been used widely to identify potentially interesting overexpressed or repressed genes in a variety of compared samples. When used in studying tissue samples, it inevitably confronts problems of limited amount of input material and cell-type heterogeneity. We report here the application of in situ hybridization as a method of confirmatory test for DD as well as definition of cell type expressing differential cDNA. This procedure employed material derived from a single case of human mammary, grade III, infiltrating ductal carcinoma, using free-hand microdissection, where we have compared gene expression profiles in invasive tumour with those in adjacent normal tissue. A total of 21 cDNAs were found to be differentially expressed between the two tissue types; 11 upregulated in the tumour sample and 10 upregulated in the normal sample. Six cDNAs were utilized as probes for in situ hybridization analysis of a further five cases of comparably staged breast cancer. One of these clones, 11AT1, which was found to be homologous to Hsc70, was shown to be overexpressed in tumour cells relative to adjacent normal stroma and to benign glandular epithelium in all five cases; an increase in expression was further confirmed at protein level by immunohistochemistry. The study demonstrated the applicability of in situ hybridization as a screening test in DD strategy for studying tissue material and a reasonable technique combination of identifying changes in gene expression associated with tumour development. [source] Strategies for identifying genes that play a role in spinal cord regenerationJOURNAL OF ANATOMY, Issue 1 2004M. Wintzer Abstract A search for genes that promote or block CNS regeneration requires numerous approaches; for example, tests can be made on individual candidate molecules. Here, however, we describe methods for comprehensive identification of genes up- and down-regulated in neurons that can and cannot regenerate after injury. One problem concerns identification of low-abundance genes out of the 30 000 or so genes expressed by neurons. Another difficulty is knowing whether a single gene or multiple genes are necessary. When microchips and subtractive differential display are used to identify genes turned on or off, the numbers are still too great to test which molecules are actually important for regeneration. Candidates are genes coding for trophic, inhibitory, receptor and extracellular matrix molecules, as well as unknown genes. A preparation useful for narrowing the search is the neonatal opossum. The spinal cord and optic nerve can regenerate after injury at 9 days but cannot at 12 days after birth. This narrow window allows genes responsible for the turning off of regeneration to be identified. As a next step, sites at which they are expressed (forebrain, midbrain, spinal cord, neurons or glia, intracellular or extracellular) must be determined. An essential step is to characterize proteins, their levels of expression, and their importance for regeneration. Comprehensive searches for molecular mechanisms represent a lengthy series of experiments that could help in devising strategies for repairing injured spinal cord. [source] Porcine ESTs detected by differential display representing possible candidates for the trait ,eye muscle area'JOURNAL OF ANIMAL BREEDING AND GENETICS, Issue 1 2000By S. Ponsuksili In order to identify ESTs which represent possible candidates for carcass traits in pigs, the differential display approach was used. F2 animals of a resource population and pure-bred German Landrace (DL) pigs were selected for the trait ,eye muscle area' in order to build up groups of three high and three low performing individuals within each population. To increase the probability that differentially expressed DNA fragments were not found due to the genetic background but due to differences in a few genes affecting the trait of interest, siblings were included in the high and in the low performing groups. RNA was isolated from M. longissimus dorsi and four ,intra-litter constrasting pools' were prepared: high performing F2, low performing F2, high performing DL and low performing DL. Differential display banding patterns were produced using (d)T11VA (V:A,C,G) and 20 arbitrary primers. Comparing the banding patterns of the four RNA pools revealed 27 nonshared bands. Here we report on the analysis of seven of these bands, including sequencing, search for homology and mapping using a somatic cell hybrid panel. Two clones showed high homology to known genes, two were homologous to an EST and a SINE sequence. Three clones did not show any homology. Differential expression was tested by semiquantitative reverse transcription,polymerase chain reaction (RT,PCR) and could be confirmed for six clones. [source] ,4 phosphoprotein interacts with EDD E3 ubiquitin ligase and poly(A)-binding proteinJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2010William J. McDonald Abstract Mammalian ,4 phosphoprotein, the homolog of yeast Tap42, is a component of the mammalian target-of-rapamycin (mTOR) pathway that regulates ribogenesis, the initiation of translation, and cell-cycle progression. ,4 is known to interact with the catalytic subunit of protein phosphatase 2A (PP2Ac) and to regulate PP2A activity. Using ,4 as bait in yeast two-hybrid screening of a human K562 erythroleukemia cDNA library, EDD (E3 isolated by differential display) E3 ubiquitin ligase was identified as a new protein partner of ,4. EDD is the mammalian ortholog of Drosophila hyperplastic discs gene (hyd) that controls cell proliferation during development. The EDD protein contains a PABC domain that is present in poly(A)-binding protein (PABP), suggesting that PABP may also interact with ,4. PABP recruits translation factors to the poly(A)-tails of mRNAs. In the present study, immunoprecipitation/immunoblotting (IP/IB) analyses showed a physical interaction between ,4 and EDD in rat Nb2 T-lymphoma and human MCF-7 breast cancer cell lines. ,4 also interacted with PABP in Nb2, MCF-7 and the human Jurkat T-leukemic and K562 myeloma cell lines. COS-1 cells, transfected with Flag-tagged-pSG5-EDD, gave a (Flag)-EDD,,4 immunocomplex. Furthermore, deletion mutants of ,4 were constructed to determine the binding site for EDD. IP/IB analysis showed that EDD bound to the C-terminal region of ,4, independent of the ,4-PP2Ac binding site. Therefore, in addition to PP2Ac, ,4 interacts with EDD and PABP, suggesting its involvement in multiple steps in the mTOR pathway that leads to translation initiation and cell-cycle progression. J. Cell. Biochem. 110: 1123,1129, 2010. Published 2010 Wiley-Liss, Inc. [source] From differential display to DNA microarrays,a personal accountJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2006Peng Liang This article is a tribute to Dr. Arthur Pardee, one of the most innovative and brilliant scientists of our time, on the occasion of his 85th birthday. In this partially perspective and partially review piece, I look back how fate, by twist and turn, has led me eventually to his lab at Harvard where we worked out the Differential Display technology from scratch, how the method has revolutionized the field of gene expression analysis and where DD is taking us in the "era" of DNA microarrays. J. Cell. Physiol. 209: 653,658, 2006. © 2006 Wiley-Liss, Inc. [source] Induction of Glycerol Phosphate Dehydrogenase Gene Expression During Seizure and AnalgesiaJOURNAL OF NEUROCHEMISTRY, Issue 4 2000Wolfgang A. Link Abstract: Using mRNA differential display, we found that the gene for NAD+ -dependent glycerol phosphate dehydrogenase (GPDH; EC 1.1.1.8) is induced in rat brain following seizure activity. Northern blot and in situ hybridization analysis confirmed the differential display results; they also showed, in a separate model of neuronal activation, that after thermal noxious stimulation of the hind-paws, a similar increase in GPDH mRNA occurs in the areas of somatotopic projection in the lumbar spinal cord. Surprisingly, administration of analgesic doses of morphine or the nonsteroidal antiinflammatory drugs aspirin, metamizol (dipyrone), and indomethacin also increased GPDH mRNA levels in rat spinal cord. The opioid receptor antagonist naloxone completely blocked morphine induction of GPDH but had no effect on GPDH induction by noxious heat stimulation or metamizol treatment, implicating different mechanisms of GPDH induction. Nevertheless, in all cases, induction of the GPDH gene requires adrenal steroids and new protein synthesis, as the induction was blocked in adrenalectomized rats and by cycloheximide treatment, respectively. Our results suggest that the induction of the GPDH gene upon peripheral noxious stimulation is related to the endogenous response to pain as it is mimicked by exogenously applied analgesic drugs. [source] Novel putative nonprotein-coding RNA gene from 11q14 displays decreased expression in brains of patients with schizophreniaJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2003Oxana O. Polesskaya Abstract A modified method of differential display was employed to identify a novel gene (named PSZA11q14), the expression of which was reduced in brains from patients with schizophrenia. Decreased expression of PSZA11q14 was identified initially in Brodmann's area (BA) 21 from a small group of patients with schizophrenia (n = 4) and normal controls (n = 6) and was confirmed subsequently using independent RT-PCR assay in BA 21, 22, and 9, and in hippocampus from a larger group of patients with schizophrenia (n = 36) and controls (n = 35). PSZA11q14 is located on chromosome 11q14, an area shown previously to co-segregate with schizophrenia and related disorders in several families. Decreased expression of PSZA11q14 in patients with schizophrenia and its location on 11q14 provide converging lines of evidence indicating that PSZA11q14 may be involved in at least some cases of schizophrenia. PSZA11q14 shows no significant homology with any known gene. It has no introns and produces two RNA transcripts of ,4.5 and ,7.0 kb. The largest open reading frame (ORF) in the PSZA11q14 transcripts may potentially encode for a short polypeptide of 71 amino acids. High frequency of rare codons, the short size of this ORF, and low homology with mouse sequences, however, indicate that PSZA11q14 may instead represent a novel member of a family of nonprotein-coding RNA genes that are not translated and that function at the RNA level. PSZA11q14 is located within the first intron of the DLG-2 gene and transcribed in the opposite direction to DLG-2. These results suggest that PSZA11q14 may be considered a candidate gene for schizophrenia acting as an antisense regulator of DLG-2, which controls assembling functional N -methyl- D -aspartate (NMDA) receptors. © 2003 Wiley-Liss, Inc. [source] Complementary DNA cloning of rat spetex-1, a spermatid-expressing gene-1, encoding a 63 kDa cytoplasmic protein of elongate spermatidsMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004Hiroshi Iida Abstract We used differential display in combination with complementary DNA (cDNA) cloning approach to isolate a novel rat gene designated as spetex-1, which had an open reading frame of 1,668-length nucleotides encoding a protein of 556 amino acids. Spetex-1 mRNA was highly expressed in testis, and weekly expressed in lung, intestine, and spleen. Spetex-1 expression in the rat testes was detected first at 3 weeks in postnatal development and continued to be detected up to adulthood. A search in the databases showed that the amino acid sequence of spetex-1 was 82% identical to that of its mouse homologue found in the databases. Both rat spetex-1 and the mouse homologue contained Ser-X (X,=,His, Arg, or Asn) repeats in the middle portion of the proteins. In situ hybridization revealed that spetex-1 mRNA was expressed in haploid spermatids of step 7,18 within the seminiferous epithelium. Immunohistochemical analysis with confocal laser-scanning microscopy demonstrated that spetex-1 protein was not expressed in spermatogonia, spermatocytes, and round spermatids in adult rat testis, but was specifically detected in the residual cytoplasm of elongate spermatids of step 15,18 as well as in residual bodies engulfed by Sertoli cells. We interpreted these data as a potential role of spetex-1 in spermatogenesis, especially in cell differentiation from late elongate spermatids to mature spermatozoa. Mol. Reprod. Dev. 68: 385,393, 2004. © 2004 Wiley-Liss, Inc. [source] Comparison by restriction fragment differential display RT‐PCR of gene expression pattern in bovine oocytes matured in the presence or absence of fetal calf serumMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2001S. Jacek Rzucidlo Abstract A novel restriction fragment differential display (RFDD) RT‐PCR has been used to compare patterns of mRNA expression in bovine oocytes matured in vitro in the presence (10%) or absence of fetal calf serum (FCS). Total RNA extracted from matured and denuded oocytes was processed using display Profile kit (Display System Biotech). RFDD RT‐PCR products were separated on 6% polyacrylamide gel and analyzed using a Storm 860 scanner. Selected bands representing potentially differentially expressed fragments were excised from the gel and re‐amplified. Re‐amplified fragments with size matched to the original fragment were cloned into the TA vector and sequenced. Initially, 10 and 15 differentially expressed fragments were isolated from oocytes matured in the presence and absence of FCS, respectively. Eight out of 10 and 10 out of 15 fragments were re‐amplified successfully as evidenced bysize similarity to the original fragments. Finally, the size of six inserts sequenced from each group matched the size of corresponding original as well as re‐amplified fragments. Sequence comparison search revealed similarity of some isolated fragments to 18s ribosomal RNA, bovine apolipoprotein A‐I, bovine mitochondrion DNA, human CGI‐79 mRNA, human Ab1‐interactor protein, and bovine satellite DNA. The other sequenced fragments may represent novel genes. We showed that RFDD RT‐PCR can be effectively applied to contrast gene expression pattern in bovine oocytes and that presence or absence of FCS during maturation interval affects gene expression pattern in matured bovine oocytes. Mol. Reprod. Dev. 59:90–96, 2001. © 2001 Wiley‐Liss, Inc. [source] Alterations in Mitochondrial and Apoptosis-regulating Gene Expression in Photodynamic Therapy-resistant Variants of HT29 Colon Carcinoma Cells,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2005Xiao Yun Shen ABSTRACT Photodynamic therapy (PDT) is a novel cancer therapy inducing irreversible photodamage to tumor tissue via photosensitizer-mediated oxidative cytotoxicity. The cellular and molecular responses associated with PDT are only partially understood. We have reported previously the generation of several photosensitizer-specific PDT-resistant cell variants of HT29 human colon adenocarcinoma cells by selecting cells from sequential PDT treatment using different photosensitizers. In this report, we describe the use of messenger RNA (mRNA) differential display to identify genes that were differentially expressed in the parental HT29 cells compared with their resistant variants. In comparison with parental HT29 cells, mRNA expression was increased in the PDT-resistant cell variants for BNIP3, estrogen receptor-binding fragmentassociated gene 9, Myh-1c, cytoplasmic dynein light chain 1, small membrane protein I and differential dependent protein. In contrast, expression in the PDT-resistant variants was downregulated for NNX3, human HepG2 3,region Mbol complementary DNA, glutamate dehydrogenase, hepatomaderived growth factor and the mitochondrial genes coding for 16S ribosomal RNA (rRNA) and nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4. The reduction for mitochondrial 16S rRNA in the PDT-resistant variants was confirmed by Northern blotting, and the elevated expression of the proapoptotic BNIP3 in the PDT-resistant variants was confirmed by Northern and Western blotting analysis. We also examined the expression of some additional apoptosis-regulating genes using Western blotting. We show an increased expression of Bcl-2 and heat shock protein 27 and a downregulation of Bax in the PDT-resistant variants. In addition, the mutant p53 levels in the parental HT29 cells were reduced substantially in the PDT-resistant variants. We suggest that the altered expression in several mitochondria1 and apoptosisregulating genes contributes to PDT resistance. [source] Cloning and identification of EDD gene from ultraviolet-irradiated HaCaT cellsPHOTODERMATOLOGY, PHOTOIMMUNOLOGY & PHOTOMEDICINE, Issue 6 2006Nishma Gupta Ultraviolet (UV) radiation is one of the most important external stimuli that affects skin by inducing cancer, inflammation and cell death. To identify the regulation of genes regulated by UV during transformation, normal human keratinocyte cell line, HaCaT, was exposed to multiple doses of UVA+B (UVA , 150,200 mJ/cm2 and UVB , 15,20 mJ/cm2× 6). Malignant transformation was confirmed by formation of colonies on soft agar and DNA methylation assay. To identify the genes involved in this process, random amplification of polymorphic DNA using RNA from unexposed and multiple exposed cells was performed after each exposure. A few up-regulated genes were identified, cloned and sequenced. One of the genes had homology to EDD (E3 identified by differential display) that was up-regulated at second exposure but was down-regulated in colony-forming cells (cells that received six or more exposures) as determined by RT-PCR. This is a progesterone-induced gene and progesterone treatment reduced the extent of colony formation on soft agar plate. It is possible that hormone therapy may have some effects on skin cancer in vivo. [source] Enhanced expression of genes for ACC synthase, ACC oxidase, and NAC protein during high-temperature-induced necrosis of young inflorescences of CymbidiumPHYSIOLOGIA PLANTARUM, Issue 3 2006Satoru Mita Growing Cymbidium under high-temperature conditions (25,30°C) results in the necrosis of young inflorescences. An increase in the evolution of ethylene was correlated with the necrosis. To study the molecular aspects of high-temperature-induced necrosis of Cymbidium floral buds, we isolated complementary DNA (cDNA) clones for proteins that are likely to be involved in the biosynthesis of ethylene during high-temperature-induced necrosis of young inflorescences, namely, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (CyACS1) and ACC oxidase (CyACO1). In addition, a cDNA (CyNAC1) encoding an NAC protein whose expression is modulated during high-temperature treatment was isolated by differential display. High levels of expression of CyACS1, CyACO1 and CyNAC1 were observed in the necrotic inflorescences of wild-type Cymbidium at high temperatures. Bud necrosis was not observed in the mericlone mutant (nhn, non,high-temperature-induced necrosis) of Cymbidium. Ethylene evolution was lower in nhn than in wild-type, but application of exogenous ACC or ethephon to the young inflorescences of nhn restored the high-temperature necrosis response. Expression of CyACS1, CyACO1 and CyNAC1 did not increase with high-temperature treatment in the nhn mutant. Expression levels of CyACS1, CyACO1 and CyNAC1 in necrotic inflorescences of nhn treated with 5.0 mM ACC were much lower than in necrotic inflorescences of wild-type at high temperatures, but CyACS1 and CyNAC1 were stimulated by ACC treatment. These results suggest that ethylene is involved in high-temperature necrosis of young inflorescences of Cymbidium and that an NAC protein may be involved in the regulatory mechanisms of genes that are regulated during necrosis. [source] Gene expression associated with N-induced shifts in resource allocation in poplarPLANT CELL & ENVIRONMENT, Issue 5 2003J. E. K. COOKE ABSTRACT Surprisingly little is known about molecular mechanisms by which nitrogen (N) availability acts to modulate the growth of forest trees. To address this issue, differential display was used in conjunction with filter-based arrays to identify 52 partial cDNA clones that were significantly regulated within days in response to limiting or luxuriant levels of NH4NO3 fertilization in Populus trichocarpa Torr. & Gray × deltoides Bartr. ex Marsh. A subset of these cDNAs also demonstrated shifts in expression patterns in stem-girdled trees, a manipulative physiology technique that disrupts phloem transport. Stem girdling also induced changes in glutamine and asparagine pools which were correlated with the observed changes in expression profiles for these genes. The identity of these genes provides insight into biochemical processes that are altered by N availability in poplar. Carbon,nitrogen interactions appear to figure prominently in the N-response. The gene expression data suggest that N availability modulates the partitioning of C and N resources into metabolic fates that have the potential to alter both wood quality and quantity, including synthesis of vegetative storage proteins, cell wall components, and terpenoids. [source] Proteomic analysis of nuclear proteins from proliferative and differentiated human colonic intestinal epithelial cellsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2004Natacha Turck Abstract Self-renewing tissues such as the intestine contain progenitor proliferating cells which subsequently differentiate. Cell proliferation and differentiation involve gene regulation processes which take place in the nucleus. A human intestinal epithelial cell line model (Caco2/TC7) which reproduces these dynamic processes has been used to perform proteomic studies on nuclear proteins. Nuclei from Caco2/TC7 cells at proliferative and differentiated stages were purified by subcellular fractionation. After two-dimensional gel electrophoresis separation and ruthenium staining, 400 protein spots were detected by image analysis. Eighty-five spots corresponding to 60 different proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry in nuclei from proliferative cells. Comparison of nuclear proteomes from proliferative or differentiated cells by differential display resulted in the identification of differentially expressed proteins such as nucleolin, hnRNP A2/B1 and hnRNP A1. By using Western blot analysis, we found that the expression and number of specific isoforms of these nuclear proteins decreased in differentiated cells. Immunocytochemistry experiments also showed that in proliferative cells nucleolin was distributed in nucleoli-like bodies. In contrast, hnRNPs A2/B1 and A1 were dispersed throughout the nucleus. This study of the nuclear proteome from intestinal epithelial cells represents the first step towards the establishment of a protein database which will be a valuable resource in future studies on the differential expression of nuclear proteins in response to physiological, pharmacological and pathological modulations. [source] Prominent expression of lysyl oxidase during mouse embryonic cardiovascular developmentTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 2 2003Takeshi Tsuda Abstract By mRNA differential display in mouse hearts, lysyl oxidase (Lox), a key enzyme catalyzing cross-links in elastin and collagens, was found to be up-regulated between embryonic days 11 (E11) and 13 (E13). This was confirmed by semiquantitative RT-PCR. We analyzed its spatio-temporal expression pattern by in situ hybridization in regard to the development of myocardial cells, endocardial cushion tissue, aortic arch vessels, and epicardium. Anat Rec Part A 270A:93,96, 2003. © 2003 Wiley-Liss, Inc. [source] Identification and characterization of pin and thrum alleles of two genes that co-segregate with the Primula S locusTHE PLANT JOURNAL, Issue 1 2007Jinhong Li Summary The study of heteromorphy in Primula over the past 140 years has established the reproductive significance of this breeding system. Plants produce either thrum or pin flowers that demonstrate reciprocal herkogamy. Thrums have short styles and produce large pollen from anthers at the mouth of the flower; pins have long styles and produce small pollen from anthers located within the corolla tube. The control of heteromorphy is orchestrated by the S locus with dominant (S) and recessive (s) alleles that comprise a co-adapted linkage group of genes. Thrum plants are heterozygous (Ss) and pin plants are homozygous (ss). Reciprocal crosses between the two forms are required for fertilization; within-morph crosses are impeded by a sporophytic self-incompatibility system. Rare recombination events within the S locus produce self-fertile homostyles. As a first step towards identifying genes located at the S locus, we used fluorescent differential display to screen for differential gene expression in pin and thrum flowers. Rather than only detecting differentially regulated genes, we identified two S locus linked genes by virtue of allelic variation between pin and thrum transcripts. Analysis of pin and thrum plants together with homostyle recombinant reveals that one gene flanks the locus, whereas the other shows complete linkage. One gene is related to Arabidopsis flower-timing genes Col9 and Col10; the other encodes a small predicted membrane protein of unknown function. Notwithstanding the diallelic behaviour of the Primula S locus, analysis of pin and thrum plants reveal three alleles for each gene: two pin and one thrum. [source] Transcriptome response of the Pacific oyster (Crassostrea gigas) to infection with Vibrio tubiashii using cDNA AFLP differential displayANIMAL GENETICS, Issue 5 2009N. Taris Summary We used qualitative complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) differential display analysis and real-time, quantitative PCR (RT-qPCR) to identify genes in the Pacific oyster Crassostrea gigas, whose transcription either changes in response to exposure to a pathogenic bacterium (Vibrio tubiashii) or varies between families known to differ in sensitivity to heat stress, before and at 12 and 36 h after bacterial exposure at a temperature of 25 °C. These conditions simulate those associated with summer mortality syndrome, a poorly understood cause of massive mortalities in cultured Pacific oysters in North America, Asia and Europe. Using 32 AFLP primer pairs, we identified 92 transcript-derived fragments that are qualitatively differentially expressed. We then cloned and sequenced 14 of these fragments, designed fragment-specific primers and quantified their transcription patterns using RT-qPCR. Most of the differences in transcription patterns between stress-tolerant and stress-sensitive families were evident before bacterial exposure, and genes that responded to bacterial exposure did so in parallel between stress-sensitive and stress-tolerant families. blast searches of sequence databases revealed that these fragments represent genes involved in immune response as well as genes related to metabolic processes. Our data support the hypothesis that family level differences in resistance to stress in Pacific oysters are largely attributable to constitutive differences in gene transcription or ,general vigour' that are detectable before and maintained after infection, rather than being due to induced responses at the transcriptome level. [source] Porcine skeletal muscle differentially expressed gene CMYA1: isolation, characterization, mapping, expression and association analysis with carcass traitsANIMAL GENETICS, Issue 3 2009X. L. Xu Summary To investigate the differences in gene expression between some obese and lean pig breeds, differential display of mRNA was employed in our previous research. One differentially expressed EST (BI596262) was further identified as the porcine cardiomyopathy associated 1 (CMYA1) gene because of its homology to the human CMYA1 gene. The full-length DNA of the porcine CMYA1 gene encompasses 9379 bp, including a complete open reading frame encoding 1839 amino acid residues, a 158-bp 5,-untranslated region and a 630-bp 3,-untranslated region. The porcine CMYA1 gene was assigned to chromosome 13 by the radiation hybrid panel (IMpRH). The porcine CMYA1 gene was expressed only in the striated muscle. Single nucleotide polymorphism (SNP) scanning in the coding region identified one synonymous mutation (c.1053C>T) and three missense mutations, c.1394A>G (p.His465Arg), c.1751A>G (p.Asp582Gly) and c.3290C>A (p.Thr1097Asp). The allele frequencies were tested among about 200 unrelated pigs from several pig breeds. Linkage mapping was further conducted with the SNP c.1751A>G (p.Asp582Gly) in a Berkshire × Yorkshire resource family and this confirmed that porcine CMYA1 is closely linked with Sw344 (distance = 2 cM, LOD score is 129.47), an interesting region harbouring a QTL for back fat thickness. Association analysis in our experimental pig population showed that different genotypes of CMYA1 gene were associated with different back fat thicknesses (P < 0.05). Our results suggest that the porcine CMYA1 gene has effects on porcine back fat deposition and further investigation will be necessary to illustrate the underlying mechanisms. [source] Different expression of Litopenaeus vannamei (Boone) haemocytes to Vibrio and abiotic particle inoculationAQUACULTURE RESEARCH, Issue 9 2005Karla Montaño-Pérez Abstract Penaeid shrimps are among the most studied crustaceans, mainly regarding their immune system, and several proteins involved in the defense against pathogens have been described. Haemocytes are very dynamic cells responsible of recognition, phagocytosis, degranulation and nodule formation, but still little is known about their gene expression. Using differential display, we found modification in haemocyte gene expression after inoculation with an abiotic particle (DEAE-Sephadex) or potential pathogenic bacteria (Vibrio alginolyticus). We also noticed that some of the newly expressed genes are exclusive from a specific treatment. Here we report that haemocytes from Litopenaeus vannamei are capable of recognizing and distinguishing different foreign materials, and respond specifically to each treatment, indicating some specificity in shrimp immune response. [source] | ||||||||||||||||||||||||||||||||||