Sample records for proliferation marker genes

  1. Peroxisome proliferator-activated receptor gamma genes polymorphism (PPARGas a marker for predisposition to sports

    Drozdovska S.B.


    Full Text Available Purpose of the work is to find the molecular-genetic markers of Pro12 Ala polymorphism of PPARG of hereditary predisposition to the manifestation of a high physical performance. During the work 122 athletes of different sports and 82 people who are not involved in sports were examined. The peculiarities of distribution of allele variants of PPARG gene in groups of athletes involved in different sports were obtained. It was found that a group of highly skilled athletes involved in sports with predominantly anaerobic nature of the energy PPARG Ala allele of the gene found in 11.1% more than the group of athletes involved in sports with mainly aerobic nature of the power supply. The existence of association between the Pro12 Ala polymorphism of PPARG gene and predisposition to various sports activities was established

  2. Stromal genes add prognostic information to proliferation and histoclinical markers: a basis for the next generation of breast cancer gene signatures.

    Dwain Mefford

    Full Text Available BACKGROUND: First-generation gene signatures that identify breast cancer patients at risk of recurrence are confined to estrogen-positive cases and are driven by genes involved in the cell cycle and proliferation. Previously we induced sets of stromal genes that are prognostic for both estrogen-positive and estrogen-negative samples. Creating risk-management tools that incorporate these stromal signatures, along with existing proliferation-based signatures and established clinicopathological measures such as lymph node status and tumor size, should better identify women at greatest risk for metastasis and death. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the strength and independence of the stromal and proliferation factors in estrogen-positive and estrogen-negative patients we constructed multivariate Cox proportional hazards models along with tree-based partitions of cancer cases for four breast cancer cohorts. Two sets of stromal genes, one consisting of DCN and FBLN1, and the other containing LAMA2, add substantial prognostic value to the proliferation signal and to clinical measures. For estrogen receptor-positive patients, the stromal-decorin set adds prognostic value independent of proliferation for three of the four datasets. For estrogen receptor-negative patients, the stromal-laminin set significantly adds prognostic value in two datasets, and marginally in a third. The stromal sets are most prognostic for the unselected population studies and may depend on the age distribution of the cohorts. CONCLUSION: The addition of stromal genes would measurably improve the performance of proliferation-based first-generation gene signatures, especially for older women. Incorporating indicators of the state of stromal cell types would mark a conceptual shift from epithelial-centric risk assessment to assessment based on the multiple cell types in the cancer-altered tissue.

  3. Evaluation of potential prognostic value of Bmi-1 gene product and selected markers of proliferation (Ki-67 and apoptosis (p53 in the neuroblastoma group of tumors

    Katarzyna Taran


    Full Text Available Introduction: Cancer in children is a very important issue in pediatrics. The least satisfactory treatment outcome occurs among patients with clinically advanced neuroblastomas. Despite much research, the biology of this tumor still remains unclear, and new prognostic factors are sought. The Bmi-1 gene product is a currently highly investigated protein which belongs to the Polycomb group (PcG and has been identified as a regulator of primary neural crest cells. It is believed that Bmi‑1 and N-myc act together and are both involved in the pathogenesis of neuroblastoma. The aim of the study was to assess the potential prognostic value of Bmi-1 protein and its relations with mechanisms of proliferation and apoptosis in the neuroblastoma group of tumors.Material/Methods: 29 formalin-fixed and paraffin-embedded neuroblastoma tissue sections were examined using mouse monoclonal antibodies anti-Bmi-1, anti-p53 and anti-Ki-67 according to the manufacturer’s instructions.Results: There were found statistically significant correlations between Bmi-1 expression and tumor histology and age of patients.Conclusions: Bmi-1 seems to be a promising marker in the neuroblastoma group of tumors whose expression correlates with widely accepted prognostic parameters. The pattern of BMI-1 expression may indicate that the examined protein is also involved in maturation processes in tumor tissue.

  4. A radial glia gene marker, fatty acid binding protein 7 (FABP7, is involved in proliferation and invasion of glioblastoma cells.

    Antonella De Rosa

    Full Text Available Glioblastoma multiforme (GBM is among the most deadly cancers. A number of studies suggest that a fraction of tumor cells with stem cell features (Glioma Stem-like Cells, GSC might be responsible for GBM recurrence and aggressiveness. GSC similarly to normal neural stem cells, can form neurospheres (NS in vitro, and seem to mirror the genetic features of the original tumor better than glioma cells growing adherently in the presence of serum. Using cDNA microarray analysis we identified a number of relevant genes for glioma biology that are differentially expressed in adherent cells and neurospheres derived from the same tumor. Fatty acid-binding protein 7 (FABP7 was identified as one of the most highly expressed genes in NS compared to their adherent counterpart. We found that down-regulation of FABP7 expression in NS by small interfering RNAs significantly reduced cell proliferation and migration. We also evaluated the potential involvement of FABP7 in response to radiotherapy, as this treatment may cause increased tumor infiltration. Migration of irradiated NS was associated to increased expression of FABP7. In agreement with this, in vivo reduced tumorigenicity of GBM cells with down-regulated expression of FABP7 was associated to decreased expression of the migration marker doublecortin. Notably, we observed that PPAR antagonists affect FABP7 expression and decrease the migration capability of NS after irradiation. As a whole, the data emphasize the role of FABP7 expression in GBM migration and provide translational hints on the timing of treatment with anti-FABP7 agents like PPAR antagonists during GBM evolution.

  5. Short-term administration of rhGH increases markers of cellular proliferation but not milk protein gene expression in normal lactating women

    Maningat, Patricia D.; Sen, Partha; Rijnkels, Monique; Hadsell, Darryl L.; Bray, Molly S.; Haymond, Morey W.


    Growth hormone is one of few pharmacologic agents known to augment milk production in humans. We hypothesized that recombinant human GH (rhGH) increases the expression of cell proliferation and milk protein synthesis genes. Sequential milk and blood samples collected over four days were obtained from five normal lactating women. Following 24 h of baseline milk and blood sampling, rhGH (0.1 mg/kg/day) was administered subcutaneously once daily for 3 days. Gene expression changes were determine...

  6. Short-term administration of rhGH increases markers of cellular proliferation, but not milk protein gene expression in normal lactating women.

    Growth hormone is one of few pharmacologic agents known to augment milk production in humans. We hypothesized that recombinant human GH (rhGH) increases the expression of cell proliferation and milk protein synthesis genes. Sequential milk and blood samples collected over four days were obtained fro...

  7. The proliferation marker thymidine kinase 1 in clinical use

    Zhou, Ji; HE, ELLEN; SKOG, SVEN


    Tumor-related biomarkers are used for the diagnosis, prognosis and monitoring of treatments and follow-up of cancer patients, although only a few are fully accepted for the detection of invisible/visible tumors in health screening. Thymidine kinase 1 (TK1), a cell cycle-dependent and thus a proliferation-related marker, has been extensively studied during the last decades, using both biochemical and immunological techniques. Therefore, TK1 is an emerging potential proliferating biomarker in o...

  8. Cell proliferation markers in the transplanted canine transmissible venereal tumor

    F.G.A. Santos


    Full Text Available Adult male mongrel dogs were subcutaneously transplanted with the canine transmissible venereal tumor (TVT on the hypogastric region. Twelve specimens of tumors were collected, half during the proliferative phase and the other half during the regressive phase. Fragments of the tumor were fixed in 10% buffered formalin and routinely processed for light microscopy. Sections of 4µm were stained by Schorr or AgNOR or either immunostained for MIB1 (Ki67. Schorr stain, AgNOR and MIB1 showed an increased proliferative activity through mitotic index, nuclear argyrophilic protein stain and cycling tumoral cells in the growing tumors, respectively. All of the three cell proliferation markers were able to distinguish the TVT in both evolution phases. MIB1 monoclonal antibody was the best in the morphologic evaluation of growth and regression of TVT. This resulted in higher values than AgNORs counting and mitotic index. MIB1 immunostaining was the most effective parameter of the proliferative activity of TVT. However, a significant correlation has been detected only between mitosis counting and AgNORs.

  9. Changes in proliferating and apoptotic markers in the oviductal magnum of chickens during sexual maturation.

    Hrabia, Anna; Leśniak-Walentyn, Agnieszka; Ocłoń, Ewa; Sechman, Andrzej


    (surface epithelium and tubular glands). In summary, the results obtained provide some evidence of changes in selected proliferation- and apoptosis-related gene expression, alterations in activity of multiple apoptotic markers, and differences in the frequency of proliferating and apoptotic markers between mucosa and stroma in the oviductal magnum during the sexual maturation. Concluding, we suggest that Bcl-2, PCNA, survivin-142, and some caspases may cooperatively orchestrate a cascade of events mainly related to the cell proliferation, apoptosis, and differentiation in the chicken oviduct over the course of its development. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Complement component 1, q subcomponent binding protein is a marker for proliferation in breast cancer.

    Scully, Olivia Jane; Yu, Yingnan; Salim, Agus; Thike, Aye Aye; Yip, George Wai-Cheong; Baeg, Gyeong Hun; Tan, Puay-Hoon; Matsumoto, Ken; Bay, Boon Huat


    Complement component 1, q subcomponent binding protein (C1QBP), is a multi-compartmental protein with higher mRNA expression reported in breast cancer tissues. This study evaluated the association between immunohistochemical expression of the C1QBP protein in breast cancer tissue microarrays (TMAs) and clinicopathological parameters, in particular tumor size. In addition, an in vitro study was conducted to substantiate the breast cancer TMA findings. Breast cancer TMAs were constructed from pathological specimens of patients diagnosed with invasive ductal carcinoma. C1QBP protein and proliferating cell nuclear antigen (PCNA) immunohistochemical analyses were subsequently performed in the TMAs. C1QBP immunostaining was detected in 131 out of 132 samples examined. The C1QBP protein was predominantly localized in the cytoplasm of the breast cancer cells. Univariate analysis revealed that a higher C1QBP protein expression was significantly associated with older patients (P = 0.001) and increased tumor size (P = 0.002). Multivariate analysis showed that C1QBP is an independent predictor of tumor size in progesterone-positive tumors. Furthermore, C1QBP was also significantly correlated with expression of PCNA, a known marker of proliferation. Inhibition of C1QBP expression was performed by transfecting C1QBP siRNA into T47D breast cancer cells, a progesterone receptor-positive breast cancer cell line. C1QBP gene expression was analyzed by real-time RT-PCR, and protein expression by Western blot. Cell proliferation assays were also performed by commercially available assays. Down-regulation of C1QBP expression significantly decreased cell proliferation and growth in T47D cells. Taken together, our findings suggest that the C1QBP protein could be a potential proliferative marker in breast cancer.

  11. Sox9 Modulates Proliferation and Expression of Osteogenic Markers of Adipose-Derived Stem Cells (ASC

    Sabine Stöckl


    Full Text Available Background: Mesenchymal stem cells (MSC are promising tools for tissue-engineering and musculoskeletal regeneration. They reside within various tissues, like adipose tissue, periosteum, synovia, muscle, dermis, blood and bone marrow, latter being the most common tissue used for MSC isolation. A promising alternative source for MSC is adipose tissue due to better availability and higher yield of MSC in comparison to bone marrow. A drawback is the yet fragmentary knowledge of adipose-derived stem cell (ASC physiology in order to make them a safe tool for in vivo application. Methods/Results: Here, we identified Sox9 as a highly expressed and crucial transcription factor in undifferentiated rat ASC (rASC. In comparison to rat bone marrow-derived stem cells (rBMSC, mRNA and protein levels of Sox9 were significantly higher in rASC. To study the role of Sox9 in detail, we silenced Sox9 with shRNA in rASC and examined proliferation, apoptosis and the expression of osteogenic differentiation markers. Our results clearly point to a difference in the expression profile of osteogenic marker genes between undifferentiated rASC and rBMSC in early passages. Sox9 silencing induced the expression of osteocalcin, Vegfα and Mmp13, and decreased rASC proliferation accompanied with an induction of p21 and cyclin D1 expression and delayed S-phase entry. Conclusions: We suggest a pro-proliferative role for Sox9 in undifferentiated rASC which may explain the higher proliferation rate of rASC compared to rBMSC. Moreover, we propose an osteogenic differentiation delaying role of Sox9 in rASC which suggests that Sox9 expression is needed to maintain rASC in an undifferentiated, proliferative state.

  12. Molecular marker genes for ectomycorrhizal symbiosis

    Shiv Hiremath; Carolyn McQuattie; Gopi Podila; Jenise. Bauman


    Mycorrhizal symbiosis is a mutually beneficial association very commonly found among most vascular plants. Formation of mycorrhiza happens only between compatible partners and predicting this is often accomplished through a trial and error process. We investigated the possibility of using expression of symbiosis specific genes as markers to predict the formation of...

  13. Sometimes it takes darkness to see the light: pitfalls in the interpretation of cell proliferation markers (Ki-67 and PCNA).

    Castilla, Carmen; McDonough, Patrick; Tumer, Gizem; Lambert, Peter C; Lambert, W Clark


    The degree of cell proliferation in a tumor is often associated with metastatic risk and mortality. Proliferating cell nuclear antigen (PCNA) and Ki-67 are proliferation markers that can be used to assess malignant potential in cutaneous lesions and pathological cell proliferation in psoriasis. These markers are elevated during periods of cell proliferation; however, they are also upregulated following UV irradiation. This upregulation may be problematic, as many skin lesions are subject to sun exposure in an everyday setting.

  14. SNP marker discovery in koala TLR genes.

    Jian Cui

    Full Text Available Toll-like receptors (TLRs play a crucial role in the early defence against invading pathogens, yet our understanding of TLRs in marsupial immunity is limited. Here, we describe the characterisation of nine TLRs from a koala immune tissue transcriptome and one TLR from a draft sequence of the koala genome and the subsequent development of an assay to study genetic diversity in these genes. We surveyed genetic diversity in 20 koalas from New South Wales, Australia and showed that one gene, TLR10 is monomorphic, while the other nine TLR genes have between two and 12 alleles. 40 SNPs (16 non-synonymous were identified across the ten TLR genes. These markers provide a springboard to future studies on innate immunity in the koala, a species under threat from two major infectious diseases.

  15. [Circadian markers and genes in bipolar disorder].

    Yeim, S; Boudebesse, C; Etain, B; Belliviera, F


    Bipolar disorder is a severe and complex multifactorial disease, characterized by alternance of acute episodes of depression and mania/hypomania, interspaced by euthymic periods. The etiological determinants of bipolar disorder yet, are still poorly understood. For the last 30 years, chronobiology is an important field of investigation to better understand the pathophysiology of bipolar disorder. We conducted a review using Medline, ISI Database, EMBase, PsyInfo up to January 2015, using the following keywords combinations: "mood disorder", "bipolar disorder", "depression", "unipolar disorder", "major depressive disorder", "affective disorder", for psychiatric conditions; and "circadian rhythms", "circadian markers", "circadian gene", "clock gene", "melatonin" for circadian rhythms. The search critera was presence of word in any field of the article. Quantitative and qualitative circadian abnormalities are associated with bipolar disorders both during acute episodes and euthymic periods, suggesting that these altered circadian rhythms may represent biological trait markers of the disorder. These circadian dysfunctions were assessed by various validated tools including polysomnography, actigraphy, sleep diaries, chronotype assessments and blood melatonin/cortisol measures. Other altered endogenous circadian activities have also been reported in bipolar patients, such as hormones secretion, core body temperature or fibroblasts activity. Moreover, these markers were also altered in healthy relatives of bipolar patients, suggesting a degree of heritability. Several genetic association studies have also showed associations between multiple circadian genes and bipolar disorder, such as CLOCK, ARTNL1, GSK3β, PER3, NPAS2, NR1D1, TIMELESS, RORA, RORB, and CSNK1ε. Thus, these circadian gene variants may contribute to the genetic susceptibility of the disease. Furthermore, the study of the clock system may help to better understand some phenotypic aspects like the

  16. Flow cytometric detection of some activation and proliferation markers in human hematopoietic cell lines.

    Glasová, M; Koníková, E; Kusenda, J; Babusíková, O


    Simultaneous surface marker/DNA, cytoplasmic/DNA or nuclear/DNA staining was used to study proliferation of hematopoietic cell lines (MOLT4, BJAB, P3HR1). Different fixation/permeabilization methods (paraformaldehyde with metanol or Tween 20 or saponin, buffered formaldehyde-acetone) were used providing optimal results of the double stainings. There was a significant increase of S phase and proliferation index (PI) of CD71+ and Ki67+ MOLT4 cells in comparison with their negative counterparts. This indicates their close connection with proliferation. Unlike that, the correlation between the expression of CD38 and S phase or PI was not significant either in MOLT4 or in P3HRI cells. For cytoplasmic markers CD3 (in MOLT4 cells) and CD22 (in BJAB cells) statistically significant (cCD3) and not significant (cCD22) correlation was demonstrated between their expression and S phase or PI. Molecular equivalents of soluble fluorescein values for CD71 were always higher than for CD38. The density of these cell surface markers in addition to the percentage of their expression is of considerable significance for their evaluation as activation or proliferation markers.

  17. Cell cycle dependent RRM2 may serve as proliferation marker and pharmaceutical target in adrenocortical cancer

    Grolmusz, Vince Kornél; Karászi, Katalin; Micsik, Tamás; Tóth, Eszter Angéla; Mészáros, Katalin; Karvaly, Gellért; Barna, Gábor; Szabó, Péter Márton; Baghy, Kornélia; Matkó, János; Kovalszky, Ilona; Tóth, Miklós; Rácz, Károly; Igaz, Péter; Patócs, Attila


    Adrenocortical cancer (ACC) is a rare, but agressive malignancy with poor prognosis. Histopathological diagnosis is challenging and pharmacological options for treatment are limited. By the comparative reanalysis of the transcriptional malignancy signature with the cell cycle dependent transcriptional program of ACC, we aimed to identify novel biomarkers which may be used in the histopathological diagnosis and for the prediction of therapeutical response of ACC. Comparative reanalysis of publicly available microarray datasets included three earlier studies comparing transcriptional differences between ACC and benign adrenocortical adenoma (ACA) and one study presenting the cell cycle dependent gene expressional program of human ACC cell line NCI-H295R. Immunohistochemical analysis was performed on ACC samples. In vitro effects of antineoplastic drugs including gemcitabine, mitotane and 9-cis-retinoic acid alone and in combination were tested in the NCI-H295R adrenocortical cell line. Upon the comparative reanalysis, ribonucleotide reductase subunit 2 (RRM2), responsible for the ribonucleotide dezoxyribonucleotide conversion during the S phase of the cell cycle has been validated as cell cycle dependently expressed. Moreover, its expression was associated with the malignancy signature, as well. Immunohistochemical analysis of RRM2 revealed a strong correlation with Ki67 index in ACC. Among the antiproliferative effects of the investigated compounds, gemcitabine showed a strong inhibition of proliferation and an increase of apoptotic events. Additionally, RRM2 has been upregulated upon gemcitabine treatment. Upon our results, RRM2 might be used as a proliferation marker in ACC. RRM2 upregulation upon gemcitabine treatment might contribute to an emerging chemoresistance against gemcitabine, which is in line with its limited therapeutical efficacy in ACC, and which should be overcome for successful clinical applications.

  18. Differential expression of alkaline phosphatase gene in proliferating primary lymphocytes and malignant lymphoid cell lines.

    Latheef, S A A; Devanabanda, Mallaiah; Sankati, Swetha; Madduri, Ramanadham


    Alkaline Phosphatase (APase) activity has been shown to be enhanced specifically in mitogen stimulated B lymphocytes committed to proliferation, but not in T lymphocytes. APase gene expression was analyzed in proliferating murine and human primary lymphocytes and human malignant cell lines using reverse transcriptase and real time PCR. In mitogen stimulated murine splenic lymphocytes, enhancement of APase activity correlated well with an increase in APase gene expression. However, in mitogen stimulated murine T lymphocytes and human PBL despite a vigorous proliferative response, no increase in APase enzyme activity or gene expression was observed. A constitutive expression of APase activity concomitant with APase gene expression was observed inhuman myeloma cell line, U266 B1. However, neither enzyme activity nor gene expression of APase were observed in human T cell lymphoma, SUPT-1. The results suggest a differential expression of APase activity and its gene in proliferating primary lymphocytes of mice and humans. The specific expression of APase activity and its gene only in human myeloma cells, but not in proliferating primary B cells can be exploited as a sensitive disease marker.

  19. Effects related to gene-gene interactions of peroxisome proliferator-activated receptor on essential hypertension



    Objective To explore the impact of the gene-gene interaction among the single nucleotide polymorphisms(SNPs) of peroxisome proliferator-activated receptorα/δ/γ on essential hypertension(EH).Methods

  20. Markers of vascular differentiation, proliferation and tissue remodeling in juvenile nasopharyngeal angiofibromas



    Juvenile nasopharingeal angiofibroma (JNA) is a histologically benign locally aggressive tumor characterized by irregular vessels embedded in a fibrous stroma. Excessive vascularity results in bleeding complications, and the inhibition of angiogenesis is a promising strategy for managing extensive JNA tumors. To better characterize the endothelial components of JNA, we aimed to evaluate markers of vascular differentiation and proliferation, such as friend leukemia integration-1 (FLI-1) and endoglin, lymphatic markers, including podoplanin and vascular endothelial growth factor receptor 3 (VEGFR3) and its cognate ligand VEGFC, GLUT-1, a diagnostic marker that discriminates between hemangiomas and vascular malformations, and two markers of tissue remodeling, stromelysin 3 (ST3) and secreted acid protein rich in cysteine (SPARC). Antigens were assessed immunohistochemically in vessels and stromal cells of JNA archival cases (n=22). JNA endothelial cells were positive for endoglin, VEGFC and FLI-1, whereas podoplanin and VEGFR3 were negative in all cases. Both endothelial cells and fibroblasts stained for ST3 and SPARC. GLUT-1 was investigated in JNA cases, in infantile hemangiomas (n=123) and in vascular malformations (n=135) as controls. JNAs and vascular malformations were GLUT-1-negative, while hemangiomas showed positive staining. The presence of markers of endothelial differentiation and proliferation highlighted the hyper-proliferative state of JNA vessels. The absence of podoplanin and VEGFR3 underscores their blood endothelial cell characteristic. The absence of GLUT-1 discriminates JNAs from hemangiomas. ST3 and SPARC up-regulation in endothelial cells and fibroblasts may contribute to a compensatory signaling for controlling angiogenesis. Some of these markers may eventually serve as therapeutic targets. Our results may aid in the understanding of JNA pathophysiology. PMID:22993619

  1. Proliferating Cellular Nuclear Antigen Expression as a Marker of Perivascular Macrophages in Simian Immunodeficiency Virus Encephalitis

    Williams, Kenneth; Schwartz, Annette; Corey, Sarah; Orandle, Marlene; Kennedy, William; Thompson, Brendon; Alvarez, Xavier; Brown, Charlie; Gartner, Suzanne; Lackner, Andrew


    Brain perivascular macrophages are a major target of simian immunodeficiency virus (SIV) infection in rhesus macaques and HIV infection in humans. Perivascular macrophages are distinct from parenchymal microglia in their location, morphology, expression of myeloid markers, and turnover in the CNS. In contrast to parenchymal microglia, perivascular macrophages are continuously repopulated by blood monocytes, which undergo maturation to macrophages on entering the central nervous system (CNS). We studied differences in monocyte/macrophages in vivo that might account for preferential infection of perivascular macrophages by SIV. In situ hybridization for SIV and proliferating cellular nuclear antigen (PCNA) immunohistochemistry demonstrated that SIV-infected and PCNA-positive cells were predominantly found in perivascular cuffs of viremic animals and in histopathological lesions that characterize SIV encephalitis (SIVE) in animals with AIDS. Multilabel techniques including double-label immunohistochemistry and combined in situ hybridization and immunofluorescence confocal microscopy revealed numerous infected perivascular macrophages that were PCNA-positive. Outside the CNS, SIV-infected, PCNA-expressing macrophage subpopulations were found in the small intestine and lung of animals with AIDS. While PCNA is used as a marker of cell proliferation it is also strongly expressed in non-dividing cells undergoing DNA synthesis and repair. Therefore, more specific markers for cell proliferation including Ki-67, topoisomerase IIα, and bromodeoxyuridine (BrdU) incorporation were used which indicated that PCNA-positive cells within SIVE lesions were not proliferating. These observations are consistent with perivascular macrophages as terminally differentiated, non-dividing cells and underscores biological differences that could potentially define mechanisms of preferential, productive infection of perivascular macrophages in the rhesus macaque model of neuroAIDS. These studies

  2. Marker2sequence, mine your QTL regions for candidate genes

    Chibon, P.Y.F.R.P.; Schoof, H.; Visser, R.G.F.; Finkers, H.J.


    Marker2sequence (M2S) aims at mining quantitative trait loci (QTLs) for candidate genes. For each gene, within the QTL region, M2S uses data integration technology to integrate putative gene function with associated gene ontology terms, proteins, pathways and literature. As a typical QTL region

  3. Chondrocyte Culture in Three Dimensional Alginate Sulfate Hydrogels Promotes Proliferation While Maintaining Expression of Chondrogenic Markers

    Mhanna, Rami; Kashyap, Aditya; Palazzolo, Gemma; Vallmajo-Martin, Queralt; Becher, Jana; Möller, Stephanie; Schnabelrauch, Matthias


    The loss of expression of chondrogenic markers during monolayer expansion remains a stumbling block for cell-based treatment of cartilage lesions. Here, we introduce sulfated alginate hydrogels as a cartilage biomimetic biomaterial that induces cell proliferation while maintaining the chondrogenic phenotype of encapsulated chondrocytes. Hydroxyl groups of alginate were converted to sulfates by incubation with sulfur trioxide–pyridine complex (SO3/pyridine), yielding a sulfated material cross-linkable with calcium chloride. Passage 3 bovine chondrocytes were encapsulated in alginate and alginate sulfate hydrogels for up to 35 days. Cell proliferation was five-fold higher in alginate sulfate compared with alginate (p=0.038). Blocking beta1 integrins in chondrocytes within alginate sulfate hydrogels significantly inhibited proliferation (p=0.002). Sulfated alginate increased the RhoA activity of chondrocytes compared with unmodified alginate, an increase that was blocked by β1 blocking antibodies (p=0.017). Expression and synthesis of type II collagen, type I collagen, and proteoglycan was not significantly affected by the encapsulation material evidenced by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. Alginate sulfate constructs showed an opaque appearance in culture, whereas the unmodified alginate samples remained translucent. In conclusion, alginate sulfate provides a three dimensional microenvironment that promotes both chondrocyte proliferation and maintenance of the chondrogenic phenotype and represents an important advance for chondrocyte-based cartilage repair therapies providing a material in which cell expansion can be done in situ. PMID:24320935

  4. Expression of Wnt/β-catenin signaling, stem-cell markers and proliferating cell markers in rat whisker hair follicles.

    Lin, Chang-min; Yuan, Yan-ping; Chen, Xian-cai; Li, Hai-hong; Cai, Bo-zhi; Liu, Yang; Zhang, Huan; Li, Yu; Huang, Keng


    The rat whisker hair follicle (HF) is a model for studying the reconstruction of the HF or dermal papilla (DP), and involves the Wnt/β-catenin signaling pathway, which is a key pathway in HF development and HF cycling after birth. It has been reported that Wnt/catenin signaling plays an indispensable role in human or rat pelages development and postnatal growth. However, the distribution of some Wnt/β-catenin signaling pathway factors and their relationship with the epithelial stem cell markers in whisker follicles has not been characterized. In this study, we investigated the immunolocalization of Wnt/catenin signaling pathway members, including Wnt10b, Wnt10a, Wnt5a, β-catenin, and downstream lymphoid enhancer-binding factor 1 (LEF1) and transcription factor 3 (TCF3), as well as, HF stem-cell markers CD34, CK15 and proliferating cell nuclear antigen (PCNA) protein, in rat anagen phase whisker follicles. β-catenin, Wnt5a, Wnt10b, Wnt10a, LEF1, and TCF3 were expressed in the outer root sheath (ORS), inner root sheath, matrix and hair shaft of anagen follicles. β-catenin, Wnt10b, LEF1, and TCF3 were highly expressed and Wnt5a and Wnt10a weakly expressed in DP and dermal sheath (DS) regions. The expression of α-smooth muscle actin was strong in the lower DS and it was also detected in some DP cells. CD34, CK15 and PCNA were all expressed in the ORS; and CD34 and PCNA were also detected in the matrix, however CD34 was extensively expressed in DP and DS regions. Our studies located the position of Wnts, downstream LEF1 and TCF3 and stem cell marker proteins, which provide new information in understanding the role of the Wnt singaling pathway in whisker follicles' growth.

  5. Cancer specificity of promoters of the genes controlling cell proliferation.

    Kashkin, Kirill; Chernov, Igor; Stukacheva, Elena; Monastyrskaya, Galina; Uspenskaya, Natalya; Kopantzev, Eugene; Sverdlov, Eugene


    Violation of proliferation control is a common feature of cancer cells. We put forward the hypothesis that promoters of genes involved in the control of cell proliferation should possess intrinsic cancer specific activity. We cloned promoter regions of CDC6, POLD1, CKS1B, MCM2, and PLK1 genes into pGL3 reporter vector and studied their ability to drive heterologous gene expression in transfected cancer cells of different origin and in normal human fibroblasts. Each promoter was cloned in short (335-800 bp) and long (up to 2.3 kb) variants to cover probable location of core and whole promoter regulatory elements. Cloned promoters were significantly more active in cancer cells than in normal fibroblasts that may indicate their cancer specificity. Both versions of CDC6 promoters were shown to be most active while the activities of others were close to that of BIRC5 gene (survivin) gene promoter. Long and short variants of each cloned promoter demonstrated very similar cancer specificity with the exception of PLK1-long promoter that was substantially more specific than its short variant and other promoters under study. The data indicate that most of the important cis-regulatory transcription elements responsible for intrinsic cancer specificity are located in short variants of the promoters under study. CDC6 short promoter may serve as a promising candidate for transcription targeted cancer gene therapy.

  6. AC-93253 triggers the downregulation of melanoma progression markers and the inhibition of melanoma cell proliferation.

    Karwaciak, Iwona; Gorzkiewicz, Michal; Ryba, Katarzyna; Dastych, Jaroslaw; Pulaski, Lukasz; Ratajewski, Marcin


    A major challenge in anti-melanoma therapy is to develop treatments that are effective for advanced melanoma patients. Unfortunately, the currently used regimens are not efficient and have unsatisfactory effects on disease progression, thus increasing the pressure to develop new, profitable drugs and to identify new molecular targets. Here, we show for the first time that AC-93253, a SIRT2 inhibitor, exerts a negative effect on the expression of a set of genes involved in the progression and chemoresistance (e.g., oncogenes, apoptosis-related genes, ABC transporter genes, and cell cycle control genes) of melanoma cells. Furthermore, melanoma cells exposed to AC-93253 and doxorubicin displayed altered biological responses, including apoptosis and proliferation, compared to cells exposed to single treatments. Taken together, we conclude that the usage of AC-93253 in combined therapy could be a promising strategy for melanoma patients.

  7. Peroxisome proliferator-activated receptor alpha target genes.

    Rakhshandehroo, Maryam; Knoch, Bianca; Müller, Michael; Kersten, Sander


    The peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPARα binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPARα governs biological processes by altering the expression of a large number of target genes. Accordingly, the specific role of PPARα is directly related to the biological function of its target genes. Here, we present an overview of the involvement of PPARα in lipid metabolism and other pathways through a detailed analysis of the different known or putative PPARα target genes. The emphasis is on gene regulation by PPARα in liver although many of the results likely apply to other organs and tissues as well.

  8. Peroxisome Proliferator-Activated Receptor Alpha Target Genes

    Maryam Rakhshandehroo


    Full Text Available The peroxisome proliferator-activated receptor alpha (PPARα is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPARα binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPARα governs biological processes by altering the expression of a large number of target genes. Accordingly, the specific role of PPARα is directly related to the biological function of its target genes. Here, we present an overview of the involvement of PPARα in lipid metabolism and other pathways through a detailed analysis of the different known or putative PPARα target genes. The emphasis is on gene regulation by PPARα in liver although many of the results likely apply to other organs and tissues as well.

  9. RNA interference mediated JAM-A gene silencing promotes human epidermal stem cell proliferation.

    Zhou, Tong; Wu, Minjuan; Guo, Xiaocan; Liu, Houqi


    The objective of the study was to explore the influence of junctional adhesion molecule A (JAM-A) gene decoration on proliferation and differentiation of human epidermal stem cells (hEpSCs). JAM-A gene and JAM-A interference gene lentivirus eukaryotic expression vectors were established. The recombinant lentivirus was introduced into hEpSCs to observe and detect viral transfection by fluorescence microscopy and Western blot, respectively. After confirmation of successful introduction of the target gene, cell growth curves were mapped out by cytometry to detect cell proliferation in different groups. The expression of hEpSCs labeled molecules was detected by immunofluorescence, and cell safety was detected by teratoma test in all groups. (1) Fluorescence microscopy showed that in the JAM-A over-expression (JAM-A(ov) EpSCs) group, the green fluorescence was mainly distributed in the cell membrane; in the JAM-A interference (JAM-A(kd) EpSCs) group and blank vector (GFP EpSCs) group, all cell bodies were luminous. Western blot showed that JAM-A protein was up-regulated in JAM-A(ov) EpSCs and down-regulated in JAM-A(kd) EpSCs. (2) Growth curves showed that hEpSCs entered the quick-growing phase 4 days after inoculation and reached the platform phase at day 7. JAM-A(ov) EpSCs proliferated more slowly than GFP EpSCs, while JAM-A(kd) EpSCs proliferated significantly faster than GFP EpSCs. (3) Immunofluorescence showed that the expression of transient amplification epidermal marker keratin 14, hEpSCs marker keratin I9 and β-integrin was down-regulated in JAM-A(kd) EpSCs group as compared to that in the GFP EpSCs group, and the expression of epidermal terminal differentiation marker K10 was negative in the JAM-A(kd) EpSCs group. There was no significant difference in the expression of specific molecules between JAM-A(ov) EpSCs and hEpSCs. (4) The result of teratoma test was negative in all groups. The proliferative ability of hEpSCs was increased markedly after down

  10. Predictive gene signatures: molecular markers distinguishing colon adenomatous polyp and carcinoma.

    Drew, Janice E; Farquharson, Andrew J; Mayer, Claus Dieter; Vase, Hollie F; Coates, Philip J; Steele, Robert J; Carey, Francis A


    Cancers exhibit abnormal molecular signatures associated with disease initiation and progression. Molecular signatures could improve cancer screening, detection, drug development and selection of appropriate drug therapies for individual patients. Typically only very small amounts of tissue are available from patients for analysis and biopsy samples exhibit broad heterogeneity that cannot be captured using a single marker. This report details application of an in-house custom designed GenomeLab System multiplex gene expression assay, the hCellMarkerPlex, to assess predictive gene signatures of normal, adenomatous polyp and carcinoma colon tissue using archived tissue bank material. The hCellMarkerPlex incorporates twenty-one gene markers: epithelial (EZR, KRT18, NOX1, SLC9A2), proliferation (PCNA, CCND1, MS4A12), differentiation (B4GANLT2, CDX1, CDX2), apoptotic (CASP3, NOX1, NTN1), fibroblast (FSP1, COL1A1), structural (ACTG2, CNN1, DES), gene transcription (HDAC1), stem cell (LGR5), endothelial (VWF) and mucin production (MUC2). Gene signatures distinguished normal, adenomatous polyp and carcinoma. Individual gene targets significantly contributing to molecular tissue types, classifier genes, were further characterised using real-time PCR, in-situ hybridisation and immunohistochemistry revealing aberrant epithelial expression of MS4A12, LGR5 CDX2, NOX1 and SLC9A2 prior to development of carcinoma. Identified gene signatures identify aberrant epithelial expression of genes prior to cancer development using in-house custom designed gene expression multiplex assays. This approach may be used to assist in objective classification of disease initiation, staging, progression and therapeutic responses using biopsy material.

  11. Predictive gene signatures: molecular markers distinguishing colon adenomatous polyp and carcinoma.

    Janice E Drew

    Full Text Available Cancers exhibit abnormal molecular signatures associated with disease initiation and progression. Molecular signatures could improve cancer screening, detection, drug development and selection of appropriate drug therapies for individual patients. Typically only very small amounts of tissue are available from patients for analysis and biopsy samples exhibit broad heterogeneity that cannot be captured using a single marker. This report details application of an in-house custom designed GenomeLab System multiplex gene expression assay, the hCellMarkerPlex, to assess predictive gene signatures of normal, adenomatous polyp and carcinoma colon tissue using archived tissue bank material. The hCellMarkerPlex incorporates twenty-one gene markers: epithelial (EZR, KRT18, NOX1, SLC9A2, proliferation (PCNA, CCND1, MS4A12, differentiation (B4GANLT2, CDX1, CDX2, apoptotic (CASP3, NOX1, NTN1, fibroblast (FSP1, COL1A1, structural (ACTG2, CNN1, DES, gene transcription (HDAC1, stem cell (LGR5, endothelial (VWF and mucin production (MUC2. Gene signatures distinguished normal, adenomatous polyp and carcinoma. Individual gene targets significantly contributing to molecular tissue types, classifier genes, were further characterised using real-time PCR, in-situ hybridisation and immunohistochemistry revealing aberrant epithelial expression of MS4A12, LGR5 CDX2, NOX1 and SLC9A2 prior to development of carcinoma. Identified gene signatures identify aberrant epithelial expression of genes prior to cancer development using in-house custom designed gene expression multiplex assays. This approach may be used to assist in objective classification of disease initiation, staging, progression and therapeutic responses using biopsy material.

  12. PCNA--a cell proliferation marker in vocal chord cancer. Part I: Premalignant laryngeal lesions.

    Pignataro, L D; Broich, G; Lavezzi, A M; Biondo, B; Ottaviani, F


    Laryngeal hyperkeratotic lesions can progress to fully developed malignant carcinoma in some cases. These premalignant lesions are proliferative disorders whose potential for further tumour progression is perhaps difficult to assess by mere histology. Immunostaining with PCNA, a protein correlated with cell proliferation, has been used to study tissue behavior in 30 cases of premalignant laryngeal vocal chord lesions treated by epithelial stripping in microlaryngoscopy, 15 of whom had no progression and 15 had recurrence and final development of full malignancy. The results showed a statistically significantly higher PCNA-index in the cases which underwent further tumour progression towards malignancy. PCNA testing may thus be suggested as a marker for tumour progression potential and help in determining clinical treatment choices.

  13. Proliferation, angiogenesis and differentiation related markers in compact and follicular-compact thyroid carcinomas in dogs

    Pessina, P.; Castillo, V.A.; César, D.; Sartore, I.; Meikle, A.


    Immunohistochemical markers (IGF-1, IGF-1R, VEGF, FGF-2, RARα and RXR) were evaluated in healthy canine thyroid glands (n=8) and in follicular-compact (n=8) and compact thyroid carcinomas (n=8). IGF-1, IGF-1R and VEGF expression was higher in fibroblasts and endothelial cells of compact carcinoma than in healthy glands (P < 0.05). Compared to follicular-compact carcinoma, compact carcinoma had higher IGF-1R expression in fibroblasts, and higher FGF-2 expression in endothelial cells (P < 0.05). RARα expression was higher in endothelial cells of compact carcinoma than in those of other groups (P < 0.05). The upregulation of these proliferation- and angiogenesis-related factors in endothelial cells and/or fibroblasts and not in follicular cells of compact carcinoma compared to healthy glands supports the relevance of stromal cells in cancer progression. PMID:28116249

  14. BABY BOOM target genes provide diverse entry points into cell proliferation and cell growth pathways.

    Passarinho, Paul; Ketelaar, Tijs; Xing, Meiqing; van Arkel, Jeroen; Maliepaard, Chris; Hendriks, Mieke Weemen; Joosen, Ronny; Lammers, Michiel; Herdies, Lydia; den Boer, Bart; van der Geest, Lonneke; Boutilier, Kim


    Ectopic expression of the Brassica napus BABY BOOM (BBM) AP2/ERF transcription factor is sufficient to induce spontaneous cell proliferation leading primarily to somatic embryogenesis, but also to organogenesis and callus formation. We used DNA microarray analysis in combination with a post-translationally regulated BBM:GR protein and cycloheximide to identify target genes that are directly activated by BBM expression in Arabidopsis seedlings. We show that BBM activated the expression of a largely uncharacterized set of genes encoding proteins with potential roles in transcription, cellular signaling, cell wall biosynthesis and targeted protein turnover. A number of the target genes have been shown to be expressed in meristems or to be involved in cell wall modifications associated with dividing/growing cells. One of the BBM target genes encodes an ADF/cofilin protein, ACTIN DEPOLYMERIZING FACTOR9 (ADF9). The consequences of BBM:GR activation on the actin cytoskeleton were followed using the GFP:FIMBRIN ACTIN BINDING DOMAIN2 (GFP:FABD) actin marker. Dexamethasone-mediated BBM:GR activation induced dramatic changes in actin organization resulting in the formation of dense actin networks with high turnover rates, a phenotype that is consistent with cells that are rapidly undergoing cytoplasmic reorganization. Together the data suggest that the BBM transcription factor activates a complex network of developmental pathways associated with cell proliferation and growth.

  15. Comparative aspects of the proliferation marker thymidine kinase 1 in human and canine tumour diseases.

    von Euler, H; Eriksson, S


    As cell proliferation is one of the hallmarks of cancer, various types of proliferation markers are used as important tools in diagnosis, prognosis, treatment decision-making and follow-up in clinical oncology. The S phase-specific protein thymidine kinase 1 (TK1) can be used in immunohistochemistry for RNA/protein expression in tissue specimens and for activity or protein/peptide levels in serum from patients. TK1 has been used mainly in haematologic malignancies in humans, but also found beneficial in canine malignancies. As the protein sequence homology is high between humans and dogs, findings in canine models will have a high comparative value in further human research as well. In the present review, we will focus on the recent results concerning TK1's S phase-correlated expression, increased serum levels of TK1 in patients with malignancies and the relevance for veterinary and comparative oncology. Finally, the benefit of recently developed specific anti-TK1 antibodies suitable for immunologic assay is discussed.

  16. Increased proliferation of explanted vascular smooth muscle cells: a marker presaging atherogenesis.

    Absher, P M; Schneider, D J; Baldor, L C; Russell, J C; Sobel, B E


    The JCR:LA-cp homozygous cp/cp corpulent rat is genetically predisposed to develop atherosclerosis evident after 9 and 18 months of age in males and females and to manifest metabolic derangements resembling those seen in type II diabetes in humans (hyperinsulinemia, insulin resistance, hyperglycemia and hypertriglyceridemia). The present study was undertaken to determine whether vascular smooth muscle cells (SMCs) explanted from vessels destined to become atherosclerotic later in life exhibit intrinsic properties ex vivo that presage atherogenesis to provide a means for evaluating promptly intervention designed to modify it. SMCs were cultured from aortic explants of JCR:LA-cp corpulent (cp/cp) and lean control (+/+) rats of 4, 5, 6, and 9 months of age. Compared with SMCs from controls, SMCs from cp/cp rats exhibited increased proliferation, higher saturation density, increased augmentation of proliferation in response to selected mitogens and greater adherence to extracellular matrix proteins. The increased proliferative activity ex vivo anteceded by several months the development of atherosclerotic lesions in vivo. Thus, it is a promising marker in assessments of the efficacy of interventions designed to retard or prevent atherosclerosis.

  17. Retinal homeobox genes and the role of cell proliferation in cavefish eye degeneration.

    Strickler, Allen G; Famuditimi, Kuburat; Jeffery, William R


    The teleost Astyanax mexicanus exhibits eyed surface dwelling (surface fish) and blind cave dwelling (cavefish) forms. Despite lacking functional eyes as adults, cavefish embryos form eye primordia, which later arrest in development, degenerate and sink into the orbit. We are comparing the expression patterns of various eye regulatory genes during surfacefish and cavefish development to determine the cause of eye degeneration. Here we examine Rx and Chx/Vsx family homeobox genes, which have a major role in cell proliferation in the vertebrate retina. We isolated and sequenced a full-length RxcDNA clone (As-Rx1) and part of a Chx/Vsx(As-Vsx2) gene, which appear to be most closely related to the zebrafish Rx1 and Alx/Vsx2 genes respectively. In situ hybridization shows that these genes have similar but non-identical expression patterns during Astyanax eye development. Expression is first detected in the optic vesicle, then throughout the presumptive retina of the optic cup, and finally in the ciliary marginal zone (CMZ), the region of the growing retina where most new retinoblasts are formed. In addition, As-Rx1 is expressed in the outer nuclear layer (ONL) of the retina, which contains the photoreceptor cells, and As-Vsx2 is expressed in the inner nuclear layer, probably in the bipolar cells. With the exception of reduced As-Rx-1 expression in the ONL, the As-Rx1 and As-Vsx2 expression patterns were unchanged in the developing retina of two different cavefish populations, suggesting that cell proliferation is not inhibited. These results were confirmed by using PCNA and BrdU markers for retinal cell division. We conclude that the CMZ is active in cell proliferation long after eye growth is diminished and is therefore not the major cause of eye degeneration.

  18. Demonstration of the proliferation marker Ki-67 in renal biopsies: correlation to clinical findings.

    Nabokov, A; Waldherr, R; Ritz, E


    Assessment of cell proliferation in renal biopsy samples is a potentially promising analytical tool to evaluate disease activity. So far no information is available on the correlation between proliferative activity in different anatomic compartments of the kidney and clinical symptoms. To elucidate this issue, we examined renal biopsy specimens from 20 patients with systemic vasculitis (15 Wegener's granulomatosis, five microscopic polyangiitis), 20 patients with immunoglobulin (Ig) A nephropathy (IgAN), 13 patients with minimal-change disease (MCD), 11 patients with tubulointerstitial nephritis, and five patients with diabetes mellitus. The streptavidin-biotin-peroxidase complex technique was applied to autoclave-pretreated, formalin-fixed, paraffin-embedded tissue sections to label different cell types with the antibody MIB1 directed against the Ki-67 antigen. Proliferation index (PI) was estimated as the number of positively stained nuclei per glomerular cross-section or per square millimeter section area. The interstitial cells were discriminated by additional staining of Ki-67-processed samples with specific immune markers. In patients with vasculitis, PI was considerably elevated in the extracapillary glomerular compartment (0.86), in proximal tubules (6.24), and in the interstitium (8.62). High proliferative activity was also noted in interstitium (3.98) and proximal tubules (1.35) of patients with IgAN. Of particular interest was the increased interstitial proliferative activity (15.0) in diabetic patients. Resident renal cells, but not infiltrating cells, seemed to constitute the majority of the proliferating cell population in the interstitium. In systemic vasculitis, clinical disease activity was significantly correlated to endocapillary (r(s) = 0.58), extracapillary (r(s) = 0.67), proximal tubular (r(s) = 0.67), and interstitial PI (r(s) = 0.61). By multiple linear regression analysis, proximal tubular PI was correlated to the presence of hematuria

  19. Changes of number of cells expressing proliferation and progenitor cell markers with age in rabbit intervertebral discs

    Miersalijiang Yasen; Qinming Fei; William C Hutton; Jian Zhang; Jian Dong; Xiaoxing Jiang; Feng Zhang


    Basic knowledge about the normal regeneration process within the intervertebral disc (IVD) is important to the understanding of the underlying biology.The presence of progenitor and stem cells in IVD has been verified.However,changes of number of progenitor and stem cells with age are still unknown.In this study,changes of cell proliferation and progenitor cell markers with age in IVD cells from rabbits of two different ages were investigated using flow cytometry,immunohistochemistry,real-time polymerase chain reaction,and western blot analysis.Proliferating cell nuclear antigen (PCNA) was chosen as a marker for proliferation,and Notch1,Jagged1,C-KIT,CD166 were chosen as stem/progenitor cell markers.Cell cycle analysis showed that cell number in the G2/M phase of the young rabbits was significantly higher than that of mature rabbits.Immunohistochemical staining demonstrated the expression of PCNA,C-KIT,CD166,Notch1,and Jagged1 in both young and mature annulus fibrosus (AF).Protein expressions of these cell markers in the young rabbits were all significantly higher than those in the mature rabbits.The expression levels of PCNA,CD166,C-KIT,Jagged1 were significantly higher in the AF,and PCNA,C-KIT in the nucleus pulposus from young rabbits than those from the mature rabbits.These findings demonstrated that both proliferation and progenitor cells exist in rabbit IVDs and the number of cells expressing proliferation and progenitor cell markers decreases with age in the rabbit IVD cells.Methods that are designed to maintain the endogenous progenitor cells and stimulate their proliferation could be successful in preventing or inhibiting degenerative disc disease.

  20. Influence of inositol hexaphosphate on the expression of selected proliferation markers in IL-1β-stimulated intestinal epithelial cells.

    Kapral, Małgorzata; Sośnicki, Stanisław; Wawszczyk, Joanna; Węglarz, Ludmiła


    The aim of the present study was to examine the influence of IP6, a naturally occurring phytochem- ical, on the expression of genes coding for proliferation markers, i.e., cyclin D1 (CCND1) and histone H3 in IL-1β-stimulated intestinal cancer cell line Caco-2. Quantification of genes expression was carried out using real time RT-QPCR technique in Caco-2 cells after treatment with IL-1β, 1 and 2.5 mM of IP6 for 3, 6 and 12 h. In separate cultures, cells were incubated with IL-1β for the indicated times. The untreated Caco-2 cells were used as the control. In a time course experiment, stimulation of cells with IL-1β only resulted in an overex- pression of both CCND1 and histone H3 mRNAs as compared with control. IP6 had no influence on IL-1β-stimulated CCND1 expression for 3 and 6 h. After 12 h, statistically significant decrease in CCND1 mRNA was observed in cells exposed to IL-1β and IP6 (1 and 2.5 mM) in relation to cells treated with IL-1β only. The levels of H3 mRNA in IL-1β-stimulated cells and cells treated with IL-1β and IP6 revealed no statistically significant differences after 3 h. IP6 at 1 and 2.5 mM enhanced IL1β-stimulated transcription of H3 gene after 6 h. Subsequently (12 h), the combination of IP6 and IL-1β decreased H3 mRNA level compared to IL1β-treated cells. In conclusion, pro-inflammatory cytokine IL-1β up-regulates CCND1 and histone H3 mRNAs expression in Caco-2 cells. These results suggest that the ability of IP6 to inhibit colon cancer cells proliferation may be mediated through downregulation of genes encoding cyclin D1 and histone H3 at the mRNA level.

  1. Study of histopathological features and proliferation markers in cases of Wilms′ tumor

    Ram Narayan Das


    Full Text Available Context: The spectrum of pediatric renal tumors is different from adult renal tumors, and Wilms′ tumor (WT forms the majority. The histological type and clinicopathological staging are the two important prognostic parameters. The role of newer prognostic factors is not clear. Aims: This study was performed to analyze the histopathological spectrum of pediatric renal tumors and to study the expression of proliferation markers (Ki-67 and p53 in WT and correlate its expression in epithelial and blastema components in different stages. Materials and Methods: Twenty-seven cases of pediatric renal tumors were collected over 2 years. Hematoxylin-eosin staining was used for diagnosis. Immunostaining was performed for Ki-67 and p53. Ki-67 proliferation index (PI and p53 expression were determined in each case and for the epithelial and blastema components separately. Statistical Analysis and Results: We had 20 cases of WT (74.1%, three cases of mesoblastic nephroma (11.1%, three cases of clear cell sarcoma (11.1% and one case of rhabdoid tumor (3.7%. It was observed that the PI of the epithelial component (57.2% was significantly higher than that of blastema (39.53% in all stages. The PI in Stage II is significantly higher than that in Stage I. Statistical analysis could not be performed in Stages III and IV due to the small number of cases. p53 expression did not show any significant difference in the epithelial and blastema components. There was also no significant difference between the stages. Conclusion: In this study, we found the differences between PI of different tissue components of WT, with the epithelial component having a higher PI, which correlated with the stage of advancement of the disease.

  2. Tagging Salt Tolerant Gene Using PCR Markers in Soybean

    GUO Bei; QIU Li-juan; SHAO Gui-hua; CHANG Ru-zhen; LIU Li-hong; XU Zhan-you; LI Xiang-hua; SUN Jian-ying


    The purpose of this study was to screen and identify PCR markers associated with salt tolerant gene in soybean( Glycine soja L. ) so that salt tolerance can be identified efficiently and accurately. Between these tolerant and sensitivity to salt and three crosses were tested in this experiment. By BSA method, two codominant PCR markers were identified through the salt tolerant (sensitive) cuitivars bulks and the salt tolerant (sensitive) individual bulks of a F2 population. There was a 600bp band in the sensitive individuals and a 700bp band or two 700bp/600bp bands in the tolerant individuals. The markers were closely linked with salt tolerant/sensitive alleles. Moreover the markers were tested in the other two F2 populations from "salt tolerant cultivar × sensitive cuitivar" and confirmed by 12 salt tolerance cultivars and 13 salt sensitive cultivars with different genetic background. It indicated that the markers (700bp and 600bp) could be applied in salt tolerant identification of the soybean germplasm resources, and markers-assisted selection in salt tolerant breeding of soybean. The markers, its obtained method and application were patented for invention in 1998.

  3. Gene markers of cellular aging in human multipotent stromal cells in culture.

    Bellayr, Ian H; Catalano, Jennifer G; Lababidi, Samir; Yang, Amy X; Lo Surdo, Jessica L; Bauer, Steven R; Puri, Raj K


    Human multipotent stromal cells (MSCs) isolated from bone marrow or other tissue sources have great potential to treat a wide range of injuries and disorders in the field of regenerative medicine and tissue engineering. In particular, MSCs have inherent characteristics to suppress the immune system and are being studied in clinical studies to prevent graft-versus-host disease. MSCs can be expanded in vitro and have potential for differentiation into multiple cell lineages. However, the impact of cell passaging on gene expression and function of the cells has not been determined. Commercially available human MSCs derived from bone marrow from six different donors, grown under identical culture conditions and harvested at cell passages 3, 5, and 7, were analyzed with gene-expression profiling by using microarray technology. The phenotype of these cells did not change as reported previously; however, a statistical analysis revealed a set of 78 significant genes that were distinguishable in expression between passages 3 and 7. None of these significant genes corresponded to the markers established by the International Society for Cellular Therapy (ISCT) for MSC identification. When the significant gene lists were analyzed through pathway analysis, these genes were involved in the top-scoring networks of cellular growth and proliferation and cellular development. A meta-analysis of the literature for significant genes revealed that the MSCs seem to be undergoing differentiation into a senescent cell type when cultured extensively. Consistent with the differences in gene expression at passage 3 and 7, MSCs exhibited a significantly greater potential for cell division at passage 3 in comparison to passage 7. Our results identified specific gene markers that distinguish aging MSCs grown in cell culture. Confirmatory studies are needed to correlate these molecular markers with biologic attributes that may facilitate the development of assays to test the quality of MSCs

  4. Helicobacter pylori and cagA gene detected by polymerase chain reaction in gastric biopsies: correlation with histological findings, proliferation and apoptosis

    Katia Ramos Moreira Leite; Elaine Darini; Flavio Canelas Canavez; Claudia Muraro de Carvalho; Cristina Aparecida Troquez da Silveira Mitteldorf; Luiz Heraldo Camara-Lopes


    CONTEXT AND OBJECTIVE: The virulence of Helicobacter pylori (HP) in gastroduodenal disease is related to pathogenicity islands (cagPAI) present in some strains. Infection with cagPAI induces IL-8 secretion, increases epithelial cell proliferation and may be important in carcinogenesis. Our objective was to detect HP and the cagA gene (cagPAI marker) by polymerase chain reaction (PCR) and to correlate these results to histological findings, epithelial cell proliferation and apoptosis. DESIGN A...

  5. Metagenomic species profiling using universal phylogenetic marker genes

    Sunagawa, Shinichi; Mende, Daniel R; Zeller, Georg;


    To quantify known and unknown microorganisms at species-level resolution using shotgun sequencing data, we developed a method that establishes metagenomic operational taxonomic units (mOTUs) based on single-copy phylogenetic marker genes. Applied to 252 human fecal samples, the method revealed...

  6. Intermediate filament genes as differentiation markers in the leech Helobdella.

    Kuo, Dian-Han; Weisblat, David A


    The intermediate filament (IF) cytoskeleton is a general feature of differentiated cells. Its molecular components, IF proteins, constitute a large family including the evolutionarily conserved nuclear lamins and the more diverse collection of cytoplasmic intermediate filament (CIF) proteins. In vertebrates, genes encoding CIFs exhibit cell/tissue type-specific expression profiles and are thus useful as differentiation markers. The expression of invertebrate CIFs, however, is not well documented. Here, we report a whole-genome survey of IF genes and their developmental expression patterns in the leech Helobdella, a lophotrochozoan model for developmental biology research. We found that, as in vertebrates, each of the leech CIF genes is expressed in a specific set of cell/tissue types. This allows us to detect earliest points of differentiation for multiple cell types in leech development and to use CIFs as molecular markers for studying cell fate specification in leech embryos. In addition, to determine the feasibility of using CIFs as universal metazoan differentiation markers, we examined phylogenetic relationships of IF genes from various species. Our results suggest that CIFs, and thus their cell/tissue-specific expression patterns, have expanded several times independently during metazoan evolution. Moreover, comparing the expression patterns of CIF orthologs between two leech species suggests that rapid evolutionary changes in the cell or tissue specificity of CIFs have occurred among leeches. Hence, CIFs are not suitable for identifying cell or tissue homology except among very closely related species, but they are nevertheless useful species-specific differentiation markers.

  7. Stathmin in pancreatic neuroendocrine neoplasms: a marker of proliferation and PI3K signaling.

    Schimmack, Simon; Taylor, Andrew; Lawrence, Ben; Schmitz-Winnenthal, Hubertus; Fischer, Lars; Büchler, Markus W; Modlin, Irvin M; Kidd, Mark; Tang, Laura H


    Chromosome 1p35-36, which encodes tumor suppressors and mitotic checkpoint control genes, is commonly altered in human malignancies. One gene at this locus, stathmin 1 (STMN1), is involved in cell cycle progression and metastasis. We hypothesized that increased STMN1 expression may play a role in pancreatic neuroendocrine neoplasm (pNEN) malignancy. We investigated stathmin copy number variation, mRNA, and protein expression using PCR-Taqman Copy Number Assays, Q-PCR, Western blot, and immunohistochemistry. A mechanistic role for stathmin in proliferation was assessed in the BON cell line under growth-restrictive conditions and siRNA silencing. Furthermore, its role in PI3K signaling pathway activation was evaluated using pharmacological inhibitors. mRNA (p = 0.0001) and protein (p stathmin inactivation (dephosphorylation p stathmin was overexpressed and associated with pathological parameters in pancreatic NENs. We postulate that STMN1 overexpression and phosphorylation result in a loss of cell cycle mitotic checkpoint control and may render tumors amenable to PI3K inhibitory therapy.

  8. Expression of the zebrafish CD133/prominin1 genes in cellular proliferation zones in the embryonic central nervous system and sensory organs.

    McGrail, Maura; Batz, Lindsey; Noack, Kristin; Pandey, Saumya; Huang, Yong; Gu, Xun; Essner, Jeffrey J


    The CD133/prominin1 gene encodes a pentamembrane glycoprotein cell surface marker that is expressed in stem cells from neuroepithelial, hematopoietic, and various organ tissues. Here we report the analysis of two zebrafish CD133/prominin1 orthologues, prominin1a and prominin1b. The expression patterns of the zebrafish prominin1a and b genes were analyzed during embryogenesis using whole mount in situ hybridization. prominin1a and b show novel complementary and overlapping patterns of expression in proliferating zones in the developing sensory organs and central nervous system. The expression patterns suggest functional conservation of the zebrafish prominin1 genes. Initial analyses of prominin1a and b in neoplastic tissue show increased expression of both genes in a subpopulation of cells in malignant peripheral nerve sheath tumors in tp53 mutants. Based on these analyses, the zebrafish prominin1 genes will be useful markers for examining proliferating cell populations in adult organs, tissues, and tumors.

  9. Proliferation and mRNA expression of absorptive villous cell markers and mineral transporters in prolactin-exposed IEC-6 intestinal crypt cells.

    Teerapornpuntakit, Jarinthorn; Wongdee, Kannikar; Thongbunchoo, Jirawan; Krishnamra, Nateetip; Charoenphandhu, Narattaphol


    During pregnancy and lactation, prolactin (PRL) enhances intestinal absorption of calcium and other minerals for fetal development and milk production. Although an enhanced absorptive efficiency is believed to mainly result from the upregulation of mineral transporters in the absorptive villous cells, some other possibilities, such as PRL-enhanced crypt cell proliferation and differentiation to increase the absorptive area, have never been ruled out. Here, we investigated cell proliferation and mRNA expression of mineral absorption-related genes in the PRL-exposed IEC-6 crypt cells. As expected, the cell proliferation was not altered by PRL. Inasmuch as the mRNA expressions of villous cell markers, including dipeptidylpeptidase-4, lactase and glucose transporter-5, were not increased, PRL was not likely to enhance crypt cell differentiation into the absorptive villous cells. In contrast to the previous findings in villous cells, PRL was found to downregulate the expression of calbindin-D(9k), claudin-3 and occludin in IEC-6 crypt cells, while having no effect on transient receptor potential vanilloid family channels-5/6, plasma membrane Ca(2+)-ATPase (PMCA)-1b and Na(+)/Ca(2+) exchanger-1 expression. In conclusion, IEC-6 crypt cells did not respond to PRL by increasing proliferation or differentiation into villous cells. The present results thus supported the previous hypothesis that PRL enhanced mineral absorption predominantly by increasing transporter expression and activity in the absorptive villous cells. Copyright © 2012 John Wiley & Sons, Ltd.

  10. Gene expression profiles of hepatic cell-type specific marker genes in progression of liver fibrosis

    Yoshiyuki Takahara; Mitsuo Takahashi; Hiroki Wagatsuma; Fumihiko Yokoya; Qing-Wei Zhang; Mutsuyo Yamaguchi; Hiroyuki Aburatani; Norifumi Kawada


    AIM: To determine the gene expression profile data for the whole liver during development of dimethylnitrosamine (DMN)-induced hepatic fibrosis.METHODS: Marker genes were identified for different types of hepatic cells, including hepatic stellate cells (HSCs), Kupffer cells (including other inflammatory cells),and hepatocytes, using independent temporal DNA microarray data obtained from isolated hepatic cells.RESULTS: The cell-type analysis of gene expression gave several key results and led to formation of three hypotheses: (1) changes in the expression of HSCspecific marker genes during fibrosis were similar to gene expression data in in vitro cultured HSCs, suggesting a major role of the self-activating characteristics of HSCs in formation of fibrosis; (2) expression of mast cell-specific marker genes reached a peak during liver fibrosis,suggesting a possible role of mast cells in formation of fibrosis; and (3) abnormal expression of hepatocytespecific marker genes was found across several metabolic pathways during fibrosis, including sulfur-containing amino acid metabolism, fatty acid metabolism, and drug metabolism, suggesting a mechanistic relationship between these abnormalities and symptoms of liver fibrosis.CONCLUSION: Analysis of marker genes for specific hepatic cell types can identify the key aspects of fibrogenesis. Sequential activation of inflammatory cells and the self-supporting properties of HSCs play an important role in development of fibrosis.

  11. Hepatic proliferation and angiogenesis markers are increased after portal deprivation in rats: a study of molecular, histological and radiological changes.

    Florent Guérin

    Full Text Available To determine the pathogenesis of liver nodules, and lesions similar to obliterative portal venopathy, observed after portosystemic shunts or portal vein thrombosis in humans.We conducted an experimental study comparing portacaval shunt (PCS, total portal vein ligation (PVL, and sham (S operated rats. Each group were either sacrificed at 6 weeks (early or 6 months (late. Arterial liver perfusion was studied in vivo using CT, and histopathological changes were noted. Liver mRNA levels were quantified by RT-QPCR for markers of inflammation (Il10, Tnfa, proliferation (Il6st, Mki67, Hgf, Hnf4a, angiogenesis: (Vegfa, Vegfr 1, 2 and 3; Pgf, oxidative stress (Nos2, and 3, Hif1a, and fibrosis (Tgfb. PCS and PVL were compared to the S group.Periportal fibrosis and arterial proliferation was observed in late PCS and PVL groups. CT imaging demonstrated increased arterial liver perfusion in the PCS group. RT-QPCR showed increased inflammatory markers in PCS and PVL early groups. Tnfa and Il10 were increased in PCS and PVL late groups respectively. All proliferative markers increased in the PCS, and Hnf4a in the PVL early groups. Mki67 and Hnf4a were increased in the PCS late group. Nos3 was increased in the early and late PCS groups, and Hif1a was decreased in the PVL groups. Markers of angiogenesis were all increased in the early PCS group, and Vegfr3 and Pgf in the late PCS group. Only Vegfr3 was increased in the PVL groups. Tgf was increased in the PCS groups.Portal deprivation in rats induces a sustained increase in intrahepatic markers of inflammation, angiogenesis, proliferation, and fibrosis.

  12. Identification of a RAPD marker linked to a blast resistance gene in Oryza sativa L.

    LUJun; ZHUANGJieyun; LINHongxuan; ZHENGKangle


    Marker-aided selection has received more attention in recent years. This relies on the exploitation of close linkage between molecular markers and target gene(s). We report here a randomly amplified polymorphic DNA (RAID) marker tightly linked to the blast resistance gene Pi-11(t) derived from Hongjiaozhan, which confers the resistante to race ZBI of Pyricularia oryzae Car.

  13. Monitoring aromatic hydrocarbon biodegradation by functional marker genes

    Nyyssoenen, Mari [Technical Research Centre of Finland, Espoo (Finland)], E-mail:; Piskonen, Reetta; Itaevaara, Merja [Technical Research Centre of Finland, Espoo (Finland)


    The development of biological treatment technologies for contaminated environments requires tools for obtaining direct information about the biodegradation of specific contaminants. The potential of functional gene array analysis to monitor changes in the amount of functional marker genes as indicators of contaminant biodegradation was investigated. A prototype functional gene array was developed for targeting key functions in the biodegradation of naphthalene, toluene and xylenes. Internal standard probe based normalization was introduced to facilitate comparison across multiple samples. Coupled with one-colour hybridization, the signal normalization improved the consistency among replicate hybridizations resulting in better discrimination for the differences in the amount of target DNA. During the naphthalene biodegradation in a PAH-contaminated soil slurry microcosm, the normalized hybridization signals in naphthalene catabolic gene probes were in good agreement with the amount of naphthalene-degradation genes and the production of {sup 14}CO{sub 2}. Gene arrays provide efficient means for monitoring of contaminant biodegradation in the environment. - Functional gene array analysis coupled with one-colour hybridization and internal standard based signal normalization provides efficient tool for monitoring contaminant biodegradation processes.

  14. The Effect of EPO Gene Overexpression on Proliferation and Migration of Mouse Bone Marrow-Derived Mesenchymal Stem Cells.

    Lin, Haihong; Luo, Xinping; Jin, Bo; Shi, Haiming; Gong, Hui


    The aim of this study is to investigate the effect of erythropoietin (EPO) gene overexpression on proliferation and migration of mouse bone marrow-derived mesenchymal stem cells (MSCs), and to determine the underlying signaling pathway. Mouse MSCs were cultured in vitro and EPO gene was transfected into the 6th generation of MSCs via lentivirus vector. The transfected cells were identified by flow cytometry and the EPO levels in supernatant were measured with ELISA. In addition, cell proliferation was assessed by CCK-8 assay and cell migration was evaluated by Transwell assay. The activation of Akt, ERK1/2, and p38MAPK signaling was detected by western blotting. The lentivirus vector containing EPO was successfully constructed and transfected into MSCs. No remarkable change was found in the cell surface markers after transfection while a significant increase of EPO level in supernatant was noticed in transfected MSCs compared to controls (P EPO modification enhanced the phosphorylation of PI3K/Akt and ERK signaling pathway, and suppressed the phosphorylation of p38MAPK without affecting the levels of total Akt, ERK1/2, and p38MAPK in MSCs. After transfection, MSCs secreted more EPO which enhanced the capability of proliferation and migration. Moreover, our results suggested that the enhanced proliferation and migration might be associated with activation of PI3K/Akt and ERK or inhibition of P38MAPK signaling pathway.

  15. Gene expression markers for Caenorhabditis elegans vulval cells.

    Inoue, Takao; Sherwood, David R; Aspöck, Gudrun; Butler, James A; Gupta, Bhagwati P; Kirouac, Martha; Wang, Minqin; Lee, Pei-Yun; Kramer, James M; Hope, Ian; Bürglin, Thomas R; Sternberg, Paul W


    The analysis of cell fate patterning during the vulval development of Caenorhabditis elegans has relied mostly on the direct observation of cell divisions and cell movements (cell lineage analysis). However, reconstruction of the developing vulva from EM serial sections has suggested seven different cell types (vulA, vulB1, vulB2, vulC, vulD, vulE, and vulF), many of which cannot be distinguished based on such observations. Here we report the vulval expression of seven genes, egl-17, cdh-3, ceh-2, zmp-1, B0034.1, T04B2.6 and F47B8.6 based on gfp, cfp and yfp (green fluorescent protein and color variants) reporter fusions. Each gene expresses in a specific subset of vulval cells, and is therefore useful as a marker for vulval cell fates. Together, expressions of markers distinguish six cell types, and reveal a strict temporal control of gene expression in the developing vulva.

  16. Production of marker-free disease-resistant potato using isopentenyl transferase gene as a positive selection marker.

    Khan, Raham Sher; Ntui, Valentine Otang; Chin, Dong Poh; Nakamura, Ikuo; Mii, Masahiro


    The use of antibiotic or herbicide resistant genes as selection markers for production of transgenic plants and their continuous presence in the final transgenics has been a serious problem for their public acceptance and commercialization. MAT (multi-auto-transformation) vector system has been one of the different strategies to excise the selection marker gene and produce marker-free transgenic plants. In the present study, ipt (isopentenyl transferase) gene was used as a selection marker gene. A chitinase gene, ChiC (isolated from Streptomyces griseus strain HUT 6037) was used as a gene of interest. ChiC gene was cloned from the binary vector, pEKH1 to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacterium tumefaciens strain EHA105. The infected tuber discs of potato were cultured on hormone- and antibiotic-free MS medium. Seven of the 35 explants infected with the pMAT21/ChiC produced shoots. The same antibiotic- and hormones-free MS medium was used in subcultures of the shoots (ipt like and normal shoots). Molecular analyses of genomic DNA from transgenic plants confirmed the integration of gene of interest and excision of the selection marker in 3 of the 7 clones. Expression of ChiC gene was confirmed by Northern blot and western blot analyses. Disease-resistant assay of the marker-free transgenic, in vitro and greenhouse-grown plants exhibited enhanced resistance against Alternaria solani (early blight), Botrytis cinerea (gray mold) and Fusarium oxysporum (Fusarium wilt). From these results it could be concluded that ipt gene can be used as a selection marker to produce marker-free disease-resistant transgenic potato plants on PGR- and antibiotic-free MS medium.

  17. Human Homolog of Drosophila Ariadne (HHARI) is a marker of cellular proliferation associated with nuclear bodies

    Elmehdawi, Fatima; Wheway, Gabrielle; Szymanska, Katarzyna [Division of Clinical Sciences, Leeds Institute of Molecular Medicine, Level 8, Wellcome Trust Brenner Building, University of Leeds, Leeds, LS9 7TF West Yorkshire (United Kingdom); Adams, Matthew [BioScreening Technology Group, Biomedical Health Research Center, Wellcome Trust Brenner Building, University of Leeds, Leeds, LS9 7TF West Yorkshire (United Kingdom); High, Alec S. [Department of Histopathology, Bexley Wing, St. James' s University Hospital, Beckett Street, Leeds, LS9 7TF West Yorkshire (United Kingdom); Johnson, Colin A., E-mail: [Division of Clinical Sciences, Leeds Institute of Molecular Medicine, Level 8, Wellcome Trust Brenner Building, University of Leeds, Leeds, LS9 7TF West Yorkshire (United Kingdom); Robinson, Philip A. [Division of Clinical Sciences, Leeds Institute of Molecular Medicine, Level 8, Wellcome Trust Brenner Building, University of Leeds, Leeds, LS9 7TF West Yorkshire (United Kingdom)


    HHARI (also known as ARIH1) is an ubiquitin-protein ligase and is the cognate of the E2, UbcH7 (UBE2L3). To establish a functional role for HHARI in cellular proliferation processes, we performed a reverse genetics screen that identified n=86/522 (16.5%) ubiquitin conjugation components that have a statistically significant effect on cell proliferation, which included HHARI as a strong hit. We then produced and validated a panel of specific antibodies that establish HHARI as both a nuclear and cytoplasmic protein that is expressed in all cell types studied. HHARI was expressed at higher levels in nuclei, and co-localized with nuclear bodies including Cajal bodies (p80 coilin, NOPP140), PML and SC35 bodies. We confirmed reduced cellular proliferation after ARIH1 knockdown with individual siRNA duplexes, in addition to significantly increased levels of apoptosis, an increased proportion of cells in G2 phase of the cell cycle, and significant reductions in total cellular RNA levels. In head and neck squamous cell carcinoma biopsies, there are higher levels of HHARI expression associated with increased levels of proliferation, compared to healthy control tissues. We demonstrate that HHARI is associated with cellular proliferation, which may be mediated through its interaction with UbcH7 and modification of proteins in nuclear bodies. -- Highlights: ► We produce and validate new antibody reagents for the ubiquitin-protein ligase HHARI. ► HHARI colocalizes with nuclear bodies including Cajal, PML and SC35 bodies. ► We establish new functions in cell proliferation regulation for HHARI. ► Increased HHARI expression associates with squamous cell carcinoma and proliferation.

  18. Markers for sebaceoma show a spectrum of cell cycle regulators, tumor suppressor genes, and oncogenes

    Ana Maria Abreu Velez


    Full Text Available Background: Sebaceoma is a tumor for which the causative oncogenes are not well-understood. Sebaceomas demonstrate some histopathologic features similar to basal cell carcinoma (BCC, such as palisading borders and basaloid cells with additional features, including foamy cytoplasm and indented nuclei. Aims: We examine multiple cell-cycle, oncogene, and tumor suppressor gene markers in sebaceomas, to try to find some suitable biological markers for this tumor, and compare with other published studies. Materials and Methods: We investigated a panel of immunohistochemical (IHC stains that are important for cellular signaling, including a cell cycle regulator, tumor suppressor gene, oncogene, hormone receptor, and genomic stability markers in our cohort of sebaceomas. We collected 30 sebaceomas from three separate USA dermatopathology laboratories. The following IHC panel: Epithelial membrane antigen (EMA/CD227, cytokeratin AE1/AE3, cyclin D1, human breast cancer 1 protein (BRCA-1, C-erb-2, Bcl-2, human androgen receptor (AR, cyclin-dependent kinase inhibitor 1B (p27 kip1 , p53, topoisomerase II alpha, proliferating cell nuclear antigen, and Ki-67 were tested in our cases. Results: EMA/CD227 was positive in the well-differentiated sebaceomas (13/30. Cyclin-dependent kinase inhibitor 1B was positive in tumors with intermediate differentiation (22/30. The less well-differentiated tumors failed to stain with EMA and AR. Most of the tumors with well-differentiated palisaded areas demonstrated positive staining for topoisomerase II alpha, p27 kip1 , and p53, with positive staining in tumoral basaloid areas (22/30. Numerous tumors were focally positive with multiple markers, indicating a significant degree of variability in the complete group. Conclusions: Oncogenes, tumor suppressor genes, cell cycle regulators, and hormone receptors are variably expressed in sebaceomas. Our results suggest that in these tumors, selected marker staining seems to correlate

  19. Gene markers in brain tumors: what the epileptologist should know.

    Ostrom, Quinn; Cohen, Mark L; Ondracek, Annie; Sloan, Andrew; Barnholtz-Sloan, Jill


    Gene markers or biomarkers can be used for diagnostic or prognostic purposes for all different types of complex disease, including brain tumors. Prognostic markers can be useful to explain differences not only in overall survival but also in response to treatment and for development of targeted therapies. Multiple genes with specific types of alterations have now been identified that are associated with improved response to chemotherapy and radiotherapy, such as O(6)-methylguanine methyltranferase (MGMT) or loss of chromosomes 1p and/or 19q. Other alterations have been identified that are associated with improved overall survival, such as mutations in isocitrate dehydrogenase 1 (IDH1) and/or isocitrate dehydrogenase 2 (IDH2) or having the glioma CpG island DNA methylator phenotype (G-CIMP). There are many biomarkers that may have relevance in brain tumor-associated epilepsy that do not respond to treatment. Given the rapidly changing landscape of high throughput "omics" technologies, there is significant potential for gaining further knowledge via integration of multiple different types of high genome-wide data. This knowledge can be translated into improved therapies and clinical outcomes for patients with brain tumors.

  20. Peroxisome Proliferator-Activated Receptor Alpha Target Genes

    Rakhshandehroo, M.; Knoch, B.; Müller, M.R.; Kersten, A.H.


    The peroxisome proliferator-activated receptor alpha (PPAR alpha) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPAR alpha serves as a molecular target for hypolip

  1. Peroxisome Proliferator-Activated Receptor Alpha Target Genes

    Rakhshandehroo, M.; Knoch, B.; Müller, M.R.; Kersten, A.H.


    The peroxisome proliferator-activated receptor alpha (PPAR alpha) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPAR alpha serves as a molecular target for

  2. Proliferation rates and gene expression profiles in human lymphoblastoid cell lines from patients with depression characterized in response to antidepressant drug therapy.

    Breitfeld, J; Scholl, C; Steffens, M; Brandenburg, K; Probst-Schendzielorz, K; Efimkina, O; Gurwitz, D; Ising, M; Holsboer, F; Lucae, S; Stingl, J C


    The current therapy success of depressive disorders remains in need of improvement due to low response rates and a delay in symptomatic improvement. Reliable functional biomarkers would be necessary to predict the individual treatment outcome. On the basis of the neurotrophic hypothesis of antidepressant's action, effects of antidepressant drugs on proliferation may serve as tentative individual markers for treatment efficacy. We studied individual differences in antidepressant drug effects on cell proliferation and gene expression in lymphoblastoid cell lines (LCLs) derived from patients treated for depression with documented clinical treatment outcome. Cell proliferation was characterized by EdU (5-ethynyl-2'-deoxyuridine) incorporation assays following a 3-week incubation with therapeutic concentrations of fluoxetine. Genome-wide expression profiling was conducted by microarrays, and candidate genes such as betacellulin-a gene involved in neuronal stem cell regeneration-were validated by quantitative real-time PCR. Ex vivo assessment of proliferation revealed large differences in fluoxetine-induced proliferation inhibition between donor LCLs, but no association with clinical response was observed. Genome-wide expression analyses followed by pathway and gene ontology analyses identified genes with different expression before vs after 21-day incubation with fluoxetine. Significant correlations between proliferation and gene expression of WNT2B, FZD7, TCF7L2, SULT4A1 and ABCB1 (all involved in neurogenesis or brain protection) were also found. Basal gene expression of SULT4A1 (P=0.029), and gene expression fold changes of WNT2B by ex vivo fluoxetine (P=0.025) correlated with clinical response and clinical remission, respectively. Thus, we identified potential gene expression biomarkers eventually being useful as baseline predictors or as longitudinal targets in antidepressant therapy.

  3. Proliferation rates and gene expression profiles in human lymphoblastoid cell lines from patients with depression characterized in response to antidepressant drug therapy

    Breitfeld, J; Scholl, C; Steffens, M; Brandenburg, K; Probst-Schendzielorz, K; Efimkina, O; Gurwitz, D; Ising, M; Holsboer, F; Lucae, S; Stingl, J C


    The current therapy success of depressive disorders remains in need of improvement due to low response rates and a delay in symptomatic improvement. Reliable functional biomarkers would be necessary to predict the individual treatment outcome. On the basis of the neurotrophic hypothesis of antidepressant's action, effects of antidepressant drugs on proliferation may serve as tentative individual markers for treatment efficacy. We studied individual differences in antidepressant drug effects on cell proliferation and gene expression in lymphoblastoid cell lines (LCLs) derived from patients treated for depression with documented clinical treatment outcome. Cell proliferation was characterized by EdU (5-ethynyl-2'-deoxyuridine) incorporation assays following a 3-week incubation with therapeutic concentrations of fluoxetine. Genome-wide expression profiling was conducted by microarrays, and candidate genes such as betacellulin—a gene involved in neuronal stem cell regeneration—were validated by quantitative real-time PCR. Ex vivo assessment of proliferation revealed large differences in fluoxetine-induced proliferation inhibition between donor LCLs, but no association with clinical response was observed. Genome-wide expression analyses followed by pathway and gene ontology analyses identified genes with different expression before vs after 21-day incubation with fluoxetine. Significant correlations between proliferation and gene expression of WNT2B, FZD7, TCF7L2, SULT4A1 and ABCB1 (all involved in neurogenesis or brain protection) were also found. Basal gene expression of SULT4A1 (P=0.029), and gene expression fold changes of WNT2B by ex vivo fluoxetine (P=0.025) correlated with clinical response and clinical remission, respectively. Thus, we identified potential gene expression biomarkers eventually being useful as baseline predictors or as longitudinal targets in antidepressant therapy. PMID:27845776

  4. MGFM: a novel tool for detection of tissue and cell specific marker genes from microarray gene expression data.

    El Amrani, Khadija; Stachelscheid, Harald; Lekschas, Fritz; Kurtz, Andreas; Andrade-Navarro, Miguel A


    Identification of marker genes associated with a specific tissue/cell type is a fundamental challenge in genetic and cell research. Marker genes are of great importance for determining cell identity, and for understanding tissue specific gene function and the molecular mechanisms underlying complex diseases. We have developed a new bioinformatics tool called MGFM (Marker Gene Finder in Microarray data) to predict marker genes from microarray gene expression data. Marker genes are identified through the grouping of samples of the same type with similar marker gene expression levels. We verified our approach using two microarray data sets from the NCBI's Gene Expression Omnibus public repository encompassing samples for similar sets of five human tissues (brain, heart, kidney, liver, and lung). Comparison with another tool for tissue-specific gene identification and validation with literature-derived established tissue markers established functionality, accuracy and simplicity of our tool. Furthermore, top ranked marker genes were experimentally validated by reverse transcriptase-polymerase chain reaction (RT-PCR). The sets of predicted marker genes associated with the five selected tissues comprised well-known genes of particular importance in these tissues. The tool is freely available from the Bioconductor web site, and it is also provided as an online application integrated into the CellFinder platform ( ). MGFM is a useful tool to predict tissue/cell type marker genes using microarray gene expression data. The implementation of the tool as an R-package as well as an application within CellFinder facilitates its use.

  5. Topoisomerase II alpha and p27; alternative markers to decide on the proliferation capacity of astrocytic tumors

    Evrim ÖZTÜRK


    Full Text Available Proliferation capacity is an important parameter which enables us to predict the prognosis of tumours. Many immunohistochemical studies were conducted to search the relation of proliferative capacity with different clinical and histological parameters. Ki67 is a well known immunohistochemical marker of proliferation and some standard values have been established for Ki67 indexes of astrocytic tumours. For this purpose, considering the roles of proteins in cell cycle, some immunohistochemical markers other than Ki67 can be suggested. In this study, expressions of topoisomerase II alpha, a nuclear protein in mitotically active cells and p27, a cylin-dependent kinase inhibitor, were correlated with the grade and Ki67 indexes of 67 astrocytomas. Topoisomerase expressions demonstrated an increase with increasing grade. It also followed a parallel curve with Ki67. On the other hand, p27 had an inverse correlation with the tumor grade. The cut-off value for topoisomerase was calculated to vary 3.5% between low and high grade tumours. No cut-off value could be obtained for p27.

  6. Dose-dependent regulation of target gene expression and cell proliferation by c-Myc levels.

    Schuhmacher, Marino; Eick, Dirk


    The proto-oncogene c-myc encodes a basic helix-loop-helix leucine zipper transcription factor (c-Myc). c-Myc plays a crucial role in cell growth and proliferation. Here, we examined how expression of c-Myc target genes and cell proliferation depend on variation of c-Myc protein levels. We show that proliferation rates, the number of cells in S-phase, and cell size increased in a dose-dependent manner in response to increasing c-Myc levels. Likewise, the mRNA levels of c-Myc responsive genes steadily increased with rising c-Myc levels. Strikingly, steady-state mRNA levels of c-Myc target genes did not saturate even at highest c-Myc concentrations. These characteristics predestine c-Myc levels as a cellular rheostat for the control and fine-tuning of cell proliferation and growth rates.

  7. Peroxisome Proliferator-Activated Receptor Alpha Target Genes

    Maryam Rakhshandehroo; Bianca Knoch; Michael Müller; Sander Kersten


    The peroxisome proliferator-activated receptor alpha (PPAR alpha) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPAR alpha serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPAR alpha binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPAR alpha governs biologi...

  8. Gene expression analysis of forskolin treated basilar papillae identifies microRNA181a as a mediator of proliferation.

    Corey S Frucht

    Full Text Available BACKGROUND: Auditory hair cells spontaneously regenerate following injury in birds but not mammals. A better understanding of the molecular events underlying hair cell regeneration in birds may allow for identification and eventually manipulation of relevant pathways in mammals to stimulate regeneration and restore hearing in deaf patients. METHODOLOGY/PRINCIPAL FINDINGS: Gene expression was profiled in forskolin treated (i.e., proliferating and quiescent control auditory epithelia of post-hatch chicks using an Affymetrix whole-genome chicken array after 24 (n = 6, 48 (n = 6, and 72 (n = 12 hours in culture. In the forskolin-treated epithelia there was significant (ptwo-fold change upregulation of many genes thought to be relevant to cell cycle control and inner ear development. Gene set enrichment analysis was performed on the data and identified myriad microRNAs that are likely to be upregulated in the regenerating tissue, including microRNA181a (miR181a, which is known to mediate proliferation in other systems. Functional experiments showed that miR181a overexpression is sufficient to stimulate proliferation within the basilar papilla, as assayed by BrdU incorporation. Further, some of the newly produced cells express the early hair cell marker myosin VI, suggesting that miR181a transfection can result in the production of new hair cells. CONCLUSIONS/SIGNIFICANCE: These studies have identified a single microRNA, miR181a, that can cause proliferation in the chicken auditory epithelium with production of new hair cells.

  9. Effect of silica and gold nanoparticles on macrophage proliferation, activation markers, cytokine production, and phagocytosis in vitro.

    Bancos, Simona; Stevens, David L; Tyner, Katherine M


    The accumulation of durable nanoparticles (NPs) in macrophages following systemic administration is well described. The ultimate biological impact of this accumulation on macrophage function, however, is not fully understood. In this study, nontoxic doses of two durable NPs, SiO2 and Au, at particle sizes of ~10 nm and 300 nm were used to evaluate the effect of bioaccumulation on macrophage function in vitro using RAW 264.7 mouse macrophage-like cells as a model system. Cell proliferation, cell cycle, cytokine production, surface marker activation, and phagocytosis responses were evaluated through a panel of assays using flow cytometry and confocal microscopy. The most dramatic change in RAW 264.7 cell function was a reduction in phagocytosis as monitored by the uptake of Escherichia coli. Cells exposed to both 10 nm Au NPs and 10 nm SiO2 NPs showed ~50% decrease in phagocytosis, while the larger NPs caused a less dramatic reduction. In addition to modifying phagocytosis profiles, 10 nm SiO2 NPs caused changes in proliferation, cell cycle, and cell morphology. Au NPs had no effect on cell cycle, cytokine production, or surface markers and caused interference in phagocytosis in the form of quenching when the assay was performed via flow cytometry. Confocal microscopy analysis was used to minimize this interference and demonstrated that both sizes of Au NPs decreased the phagocytosis of E. coli. Overall, our results demonstrate that Au and SiO2 NP uptake by macrophages can influence macrophage phagocytosis in vitro without altering surface markers and cytokine production in vitro. While the biological impact of these findings remains unclear, our results indicate that bioaccumulation of durable NPs within the macrophages may lead to a suppression of bacterial uptake and possibly impair bactericidal activity.

  10. Platelet-rich plasma stimulated by pulse electric fields: Platelet activation, procoagulant markers, growth factor release and cell proliferation.

    Frelinger, A L; Torres, A S; Caiafa, A; Morton, C A; Berny-Lang, M A; Gerrits, A J; Carmichael, S L; Neculaes, V B; Michelson, A D


    Therapeutic use of activated platelet-rich plasma (PRP) has been explored for wound healing, hemostasis and antimicrobial wound applications. Pulse electric field (PEF) stimulation may provide more consistent platelet activation and avoid complications associated with the addition of bovine thrombin, the current state of the art ex vivo activator of therapeutic PRP. The aim of this study was to compare the ability of PEF, bovine thrombin and thrombin receptor activating peptide (TRAP) to activate human PRP, release growth factors and induce cell proliferation in vitro. Human PRP was prepared in the Harvest SmartPreP2 System and treated with vehicle, PEF, bovine thrombin, TRAP or Triton X-100. Platelet activation and procoagulant markers and microparticle generation were measured by flow cytometry. Released growth factors were measured by ELISA. The releasates were tested for their ability to stimulate proliferation of human epithelial cells in culture. PEF produced more platelet-derived microparticles, P-selectin-positive particles and procoagulant annexin V-positive particles than bovine thrombin or TRAP. These differences were associated with higher levels of released epidermal growth factor after PEF than after bovine thrombin or TRAP but similar levels of platelet-derived, vascular-endothelial, and basic fibroblast growth factors, and platelet factor 4. Supernatant from PEF-treated platelets significantly increased cell proliferation compared to plasma. In conclusion, PEF treatment of fresh PRP results in generation of microparticles, exposure of prothrombotic platelet surfaces, differential release of growth factors compared to bovine thrombin and TRAP and significant cell proliferation. These results, together with PEF's inherent advantages, suggest that PEF may be a superior alternative to bovine thrombin activation of PRP for therapeutic applications.

  11. Utility of the pat gene as a selectable marker gene in production of transgenic Dunaliella salina

    Hyo Sun Jung


    Full Text Available Abstract Background The objective of this study was to develop an efficient selectable marker for transgenic Dunaliella salina. Results Tests of the sensitivity of D. salina to the antibiotic chloramphenicol and the herbicide Basta® showed that cells (1.0 × 106 cells/ml treated with 1000 or 1500 μg/ml chloramphenicol died in 8 or 6 days, respectively, whereas D. salina cells (1.0 × 106 cells/ml treated with 5, 10, 20, or 40 μg/ml Basta® died in 2 days. Therefore, D. salina is more sensitive to Basta® than to chloramphenicol. To examine the possibility of using the phosphinothricin N-acetyltransferase (pat gene as a selectable marker gene, we introduced the pat genes into D. salina with particle bombardment system under the condition of helium pressure of 900 psi from a distance of 3 cm. PCR analysis confirmed that the gene was stably inserted into the cells and that the cells survived in 5 μg/ml Basta®, the medium used to select the transformed cells. Conclusions The findings of this study suggest that the pat gene can be used as an efficient selectable marker when producing transgenic D. salina.

  12. HBV X Gene Transfection Upregulates IL-1β and IL-6 Gene Expression and Induces Rat Glomerular Mesangial Cell Proliferation

    Hongzhu LU; Jianhua ZHOU


    The X gene of HBV encodes a 17-KD protein, termed HBx, which has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements. The aim of this study was to investigate the effect of HBx on gene expression of interleukin (IL)-1β and IL-6, and proliferation of rat mesangial cells in vitro. The X gene of HBV was amplified by PCR assay, and inserted into the eukaryotic expression vector pCI-neo. The structure of recombinant pCI-neo-X plasmid was proved by restrict endonuclease digestion and sequencing analysis. pCI-neo-X was transfected into cultured rat mesangial cell line in vitro via liposome. HBx expression in transfected mesangial cells was detected by Western blot. The IL-1β and IL-6 mRNA expression in those cells was assayed by semiquantitative RT-PCR. Mesangial cell proliferation was tested by MTT. The results showed that HBx was obviously expressed in cultured mesangial cell line at 36th and 48th h after transfection. The expression of IL-1β and IL-6 mRNA was simultaneously increased. The cell proliferation was also obvious at the same time. It was concluded that HBx gene transfection could induce IL-1β and IL-6 gene expression and mesangial cell proliferation. HBx may play a critical role in mesangial cell proliferation through upregulation of the IL-1β and IL-6 gene expression.

  13. Proliferating Cellular Nuclear Antigen Expression as a Marker of Perivascular Macrophages in Simian Immunodeficiency Virus Encephalitis


    Brain perivascular macrophages are a major target of simian immunodeficiency virus (SIV) infection in rhesus macaques and HIV infection in humans. Perivascular macrophages are distinct from parenchymal microglia in their location, morphology, expression of myeloid markers, and turnover in the CNS. In contrast to parenchymal microglia, perivascular macrophages are continuously repopulated by blood monocytes, which undergo maturation to macrophages on entering the central nervous system (CNS). ...

  14. Wnt target genes identified by DNA microarrays in immature CD34+ thymocytes regulate proliferation and cell adhesion

    F.J.T. Staal (Frank); F. Weerkamp (Floor); M.R.M. Baert (Miranda); C.M. van den Burg (Caroline); M. van Noort (Mascha); E.F. de Haas (Edwin); J.J.M. van Dongen (Jacques)


    textabstractThe thymus is seeded by very small numbers of progenitor cells that undergo massive proliferation before differentiation and rearrangement of TCR genes occurs. Various signals mediate proliferation and differentiation of these cells, including Wnt signals. Wnt signals i


    Jacobo de la Cuesta-Zuluaga


    Full Text Available Next generation sequencing technologies have found a widespread use in the study of host–microbe interactions due to the increase in their throughput and their ever-decreasing costs. The analysis of human-associated microbial communities using a marker gene, particularly the 16S rRNA, has greatly benefited from these technologies, the human gut microbiome research being a remarkable example of such analysis that has greatly expanded our understanding of microbe-mediated human health and disease, metabolism and food absorption. 16S studies go through a series of in vitro and in silico steps that can greatly influence their outcomes. However, the lack of a standardized workflow has led to uncertainties regarding the transparency and reproducibility of gut microbiome studies. We here discuss the most common challenges in the archetypical 16S rRNA workflow, including the extraction of total DNA, its use as template in PCR with primers that amplify specific hypervariable regions of the gene, amplicon sequencing, the denoising and removal of low quality reads, the detection and removal of chimeric sequences, the clustering of high-quality sequences into operational taxonomic units (OTUs and their taxonomic classification. We recommend the essential technical information that should be conveyed in publications for reproducibility of results and encourage non-experts to include procedures and available tools that mitigate most of the problems encountered in microbiome analysis.

  16. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng, E-mail:


    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

  17. Simplet controls cell proliferation and gene transcription during zebrafish caudal fin regeneration.

    Kizil, Caghan; Otto, Georg W; Geisler, Robert; Nüsslein-Volhard, Christiane; Antos, Christopher L


    Two hallmarks of vertebrate epimorphic regeneration are a significant increase in the proliferation of normally quiescent cells and a re-activation of genes that are active during embryonic development. It is unclear what the molecular determinants are that regulate these events and how they are coordinated. Zebrafish have the ability to regenerate several compound structures by regulating cell proliferation and gene transcription. We report that fam53b/simplet (smp) regulates both cell proliferation and the transcription of specific genes. In situ hybridization and quantitative RT-PCR experiments showed that amputation of zebrafish hearts and fins resulted in strong up-regulation of the smp gene. In regenerating adult fin, smp expression remained strong in the distal mesenchyme which later expanded to the basal layers of the distal epidermis and distal tip epithelium. Morpholino knockdown of smp reduced regenerative outgrowth by decreasing cell proliferation as measured by BrdU incorporation and histone H3 phosphorylation. In addition, smp knockdown increased the expression of msxb, msxc, and shh, as well as the later formation of ectopic bone. Taken together, these data indicate a requirement for smp in fin regeneration through control of cell proliferation, the regulation of specific genes and proper bone patterning.

  18. Metformin does not reduce markers of cell proliferation in esophageal tissues of patients with Barrett's esophagus.

    Chak, Amitabh; Buttar, Navtej S; Foster, Nathan R; Seisler, Drew K; Marcon, Norman E; Schoen, Robert; Cruz-Correa, Marcia R; Falk, Gary W; Sharma, Prateek; Hur, Chin; Katzka, David A; Rodriguez, Luz M; Richmond, Ellen; Sharma, Anamay N; Smyrk, Thomas C; Mandrekar, Sumithra J; Limburg, Paul J


    Obesity is associated with neoplasia, possibly via insulin-mediated cell pathways that affect cell proliferation. Metformin has been proposed to protect against obesity-associated cancers by decreasing serum insulin. We conducted a randomized, double-blind, placebo-controlled, phase 2 study of patients with Barrett's esophagus (BE) to assess the effect of metformin on phosphorylated S6 kinase (pS6K1), a biomarker of insulin pathway activation. Seventy-four subjects with BE (mean age, 58.7 years; 58 men [78%; 52 with BE >2 cm [70%]) were recruited through 8 participating organizations of the Cancer Prevention Network. Participants were randomly assigned to groups given metformin daily (increasing to 2000 mg/day by week 4, n = 38) or placebo (n = 36) for 12 weeks. Biopsy specimens were collected at baseline and at week 12 via esophagogastroduodenoscopy. We calculated and compared percent changes in median levels of pS6K1 between subjects given metformin vs placebo as the primary end point. The percent change in median level of pS6K1 did not differ significantly between groups (1.4% among subjects given metformin vs -14.7% among subjects given placebo; 1-sided P = .80). Metformin was associated with an almost significant reduction in serum levels of insulin (median -4.7% among subjects given metformin vs 23.6% increase among those given placebo, P = .08) as well as in homeostatic model assessments of insulin resistance (median -7.2% among subjects given metformin vs 38% increase among those given placebo, P = .06). Metformin had no effects on cell proliferation (on the basis of assays for KI67) or apoptosis (on the basis of levels of caspase 3). In a chemoprevention trial of patients with BE, daily administration of metformin for 12 weeks, compared with placebo, did not cause major reductions in esophageal levels of pS6K1. Although metformin reduced serum levels of insulin and insulin resistance, it did not discernibly alter epithelial proliferation or apoptosis in

  19. The Fto Gene Regulates the Proliferation and Differentiation of Pre-Adipocytes in Vitro

    Yang Jiao


    Full Text Available The highly regulated differentiation and proliferation of pre-adipocytes play a key role in the initiation of obesity. Fat mass and obesity associated (FTO is a novel gene strongly associated with the risk of obesity. A deficiency of FTO may cause growth retardation in addition to fat mass and adipocyte size reduction in vivo. To investigate the potential role of Fto gene on the proliferation and differentiation of pre-adipocytes, we generated Fto-knockdown and overexpressed 3T3-L1 cells. Using numerous proliferation assays our results suggest that Fto knockdown leads to suppression of proliferation, lower mitochondrial membrane potential, less cellular ATP, and decreased and smaller intracellular lipid droplets compared with controls (p < 0.05. Western blot analysis demonstrated that Fto knockdown can significantly suppress peroxisome proliferator-activated receptor gamma (PPARγ and glucose transporter type 4 (GLUT4 expression and inhibit Akt phosphorylation. By contrast, overexpression of Fto had the opposing effect on proliferation, mitochondrial membrane potential, ATP generation, in vitro differentiation, Akt phosphorylation, and PPARγ and GLUT4 expression. Moreover, we demonstrated that Wortmannin, a phosphoinositide 3-kinase (PI3K inhibitor, could inhibit phospho-Akt in Fto overexpressed 3T3-L1 cells. Taken together, the results suggest that Fto regulates the proliferation and differentiation of 3T3-L1 cells via multiple mechanisms, including PPARγ and PI3K/Akt signaling.

  20. A Chinese Herbal Decoction, Danggui Buxue Tang, Stimulates Proliferation, Differentiation and Gene Expression of Cultured Osteosarcoma Cells: Genomic Approach to Reveal Specific Gene Activation

    Roy C. Y. Choi


    Full Text Available Danggui Buxue Tang (DBT, a Chinese herbal decoction used to treat ailments in women, contains Radix Astragali (Huangqi; RA and Radix Angelicae Sinensis (Danggui; RAS. When DBT was applied onto cultured MG-63 cells, an increase of cell proliferation and differentiation of MG-63 cell were revealed: both of these effects were significantly higher in DBT than RA or RAS extract. To search for the biological markers that are specifically regulated by DBT, DNA microarray was used to reveal the gene expression profiling of DBT in MG-63 cells as compared to that of RA- or RAS-treated cells. Amongst 883 DBT-regulated genes, 403 of them are specifically regulated by DBT treatment, including CCL-2, CCL-7, CCL-8, and galectin-9. The signaling cascade of this DBT-regulated gene expression was also elucidated in cultured MG-63 cells. The current results reveal the potential usage of this herbal decoction in treating osteoporosis and suggest the uniqueness of Chinese herbal decoction that requires a well-defined formulation. The DBT-regulated genes in the culture could serve as biological responsive markers for quality assurance of the herbal preparation.


    浦佩玉; 夏之柏; 黄强; 王春艳; 王广秀


    Objective: To evaluate the effect of Cx43 gene on gap junction intercellular communication (GJIC) and proliferation of glioma cells. Methods: Cx43 cDNA was transfected into TJ905 human glioblastoma cells using lipofectamine. The expression of Cx43 was identified by Northern blot analyses, in situ hybridization and immunohistochemistry. MTT assay and average number of AgNORs (Argyrophlic nuclear organizer regions) were used to determine the cell proliferation. TUNEL method was used for detection of cell apoptosis, and scrape loading and dye tranfer method for examination of GJIC. Results: The Cx43 expression was greatly upregulated when Cx43 gene was transfected into TJ905 glioma cells. The cell proliferation was inhibited while the cell apoptosis was not increased and GJIC was significantly restored in the glioma cells tranfected with Cx43 gene. Conclusion: Cx43 gene has an inhibitory effect on the glioma cell proliferation, but no effect on induction of cell apoptosis. The restoration of GJIC may be the major mechanism involved in its effect. Cx43 gene can be the candidate for gene therapy of gliomas.

  2. Identification of Specific RAPD Markers Linked to Anthracnose Resistant Gene in Native Wild Grapes of China

    WANG Xi-ping; WANG Yue-jin; ZHOU Peng; ZHENG Xue-qin


    Randomly amplified polymorphic DNA (RAPD) was employed to detect molecular markers linked to anthracnose ( Spheceloma ampelinum de Bary) resistant gene in the native wild grapes ( Vitis L. ) of China. RAPD marker OPJ13-300 was linked to anthracnose resistant gene using 90-3 cross F1 V. quinquangularis Rehd (shang-24) × V. vinifera (Longyan). The marker was verified in 90-3 cross F1, Chinese wild grapes and V. riparia and European grape cuitivars. This work has provided a solid basis for molecular marker-assisted selection (MAS) to disease resistance and cloning of disease resistant genes.

  3. Genetic transformation of apple (Malus x domestica) without use of a selectable marker gene

    Selectable marker genes are widely used for the efficient transformation of crop plants. In most cases, antibiotic or herbicide resistance marker genes are preferred, because they tend to be most efficient. Due mainly to consumer and grower concerns, considerable effort is being put into developin...

  4. BAP1 (BRCA1-associated protein 1) is a highly specific marker for differentiating mesothelioma from reactive mesothelial proliferations.

    Cigognetti, Marta; Lonardi, Silvia; Fisogni, Simona; Balzarini, Piera; Pellegrini, Vilma; Tironi, Andrea; Bercich, Luisa; Bugatti, Mattia; Rossi, Giulio; Murer, Bruno; Barbareschi, Mattia; Giuliani, Silvia; Cavazza, Alberto; Marchetti, Gianpietro; Vermi, William; Facchetti, Fabio


    The distinction between malignant mesothelioma and reactive mesothelial proliferation can be challenging both on histology and cytology. Recently, variants of the BRCA1-associated protein 1 (BAP1) gene resulting in nuclear protein loss were reported in hereditary and sporadic mesothelioma. Using immunohistochemistry, we evaluated the utility of BAP1 expression in the differential diagnosis between mesothelioma and other mesothelial proliferations on a large series of biopsies that included 212 mesotheliomas, 12 benign mesothelial tumors, and 42 reactive mesothelial proliferations. BAP1 stain was also performed in 70 cytological samples (45 mesotheliomas and 25 reactive mesothelial proliferations). BAP1 was expressed in all benign mesothelial tumors, whereas 139/212 (66%) mesotheliomas were BAP1 negative, especially in epithelioid/biphasic compared with sarcomatoid/desmoplastic subtypes (69% vs 15%). BAP1 loss was homogeneous in neoplastic cells except for two epithelioid mesotheliomas showing tumor heterogeneity. By fluorescence in situ hybridization, BAP1 protein loss was paralleled by homozygous deletion of the BAP1 locus in the vast majority of BAP1-negative tumors (31/41, 76%), whereas 9/10 BAP1-positive mesotheliomas were normal. In biopsies interpreted as reactive mesothelial proliferation BAP1 loss was 100% predictive of malignancy, as all 6 cases subsequently developed BAP1-negative mesothelioma, whereas only 3/36 (8%) BAP1-positive cases progressed to mesothelioma. On cytology/cell blocks, benign mesothelial cells were invariably positive for BAP1, whereas 64% of mesotheliomas showed loss of protein; all 6 cases showing BAP1 negativity were associated with histological diagnosis of BAP1-negative mesothelioma. BAP1 stain also showed utility in the differential of mesothelioma from most common pleural and peritoneal mimickers, such as lung and ovary carcinomas, with specificity and sensitivity of 99/70% and 100/70%, respectively. Our results show that BAP1

  5. Oral pathogens change proliferation properties of oral tumor cells by affecting gene expression of human defensins.

    Hoppe, T; Kraus, D; Novak, N; Probstmeier, R; Frentzen, M; Wenghoefer, M; Jepsen, S; Winter, J


    The impact of oral pathogens onto the generation and variability of oral tumors has only recently been investigated. To get further insights, oral cancer cells were treated with pathogens and additionally, as a result of this bacterial cellular infection, with human defensins, which are as anti-microbial peptide members of the innate immune system. After cell stimulation, proliferation behavior, expression analysis of oncogenic relevant defensin genes, and effects on EGFR signaling were investigated. The expression of oncogenic relevant anti-microbial peptides was analyzed with real-time PCR and immunohistochemistry. Cell culture experiments were performed to examine cellular impacts caused by stimulation, i.e., altered gene expression, proliferation rate, and EGF receptor-dependent signaling. Incubation of oral tumor cells with an oral pathogen (Porphyromonas gingivalis) and human α-defensins led to an increase in cell proliferation. In contrast, another oral bacterium used, Aggregatibacter actinomycetemcomitans, enhanced cell death. The bacteria and anti-microbial peptides exhibited diverse effects on the transcript levels of oncogenic relevant defensin genes and epidermal growth factor receptor signaling. These two oral pathogens exhibited opposite primary effects on the proliferation behavior of oral tumor cells. Nevertheless, both microbe species led to similar secondary impacts on the proliferation rate by modifying expression levels of oncogenic relevant α-defensin genes. In this respect, oral pathogens exerted multiplying effects on tumor cell proliferation. Additionally, human defensins were shown to differently influence epidermal growth factor receptor signaling, supporting the hypothesis that these anti-microbial peptides serve as ligands of EGFR, thus modifying the proliferation behavior of oral tumor cells.

  6. Transcriptional Activity of HTLV-I Tax Influences the Expression of Marker Genes Associated with Cellular Transformation

    Francene J. Lemoine


    Full Text Available Human T cell leukemia virus type I (HTLV-I has been identified as the etiologic agent of adult T cell leukemia (ATL. HTLV-I encodes a transcriptional regulatory protein, Tax, which also functions as the viral transforming protein. Through interactions with a number of cellular transcription factors Tax can modulate cellular gene expression. Since the majority of Tax-responsive cellular genes are important regulators of cellular proliferation, the transactivating functions of Tax appear to be necessary for cellular transformation by HTLV-I. Gaining a complete understanding of the broad range of genes regulated by Tax, the temporal pattern of their expression, and their effects on cell function may identify early markers of disease progression mediated by this virus.

  7. Development of Molecular Marker Linked to Cf-10 Gene Using SSR and AFLP Method in Tomato

    Li Ning; Jiang Jing-bin; Li Jing-fu; Xu Xiang-yang


    The leaf mould resistance gene Cf-10 on tomato confered resistant or immune to all prevalent physiological races of Cladosporium fulvum presented in three northeastern provinces of China in inoculation test. In order to better utilize Cf-10 gene in a marker-assisted selection program and to permit the pyramiding of one or several resistance genes in a cultivar, tightly linked SSR and AFLP markers were obtained by the bulked segregant analysis method. One SSR marker and three AFLP markers were identified linked to Cf-10 gene, with the distance of 9.73, 5.8, 8.5, and 10.6 cM, respectively. These markers will facilitate the selection of resistant tomato germplasm containing Cf-10 gene.

  8. A thiostrepton resistance gene and its mutants serve as selectable markers in Geobacillus kaustophilus HTA426.

    Wada, Keisuke; Kobayashi, Jyumpei; Furukawa, Megumi; Doi, Katsumi; Ohshiro, Takashi; Suzuki, Hirokazu


    Effective utilization of microbes often requires complex genetic modification using multiple antibiotic resistance markers. Because a few markers have been used in Geobacillus spp., the present study was designed to identify a new marker for these thermophiles. We explored antibiotic resistance genes functional in Geobacillus kaustophilus HTA426 and identified a thiostrepton resistance gene (tsr) effective at 50 °C. The tsr gene was further used to generate the mutant tsr(H258Y) functional at 55 °C. Higher functional temperature of the mutant was attributable to the increase in thermostability of the gene product because recombinant protein produced from tsr(H258Y) was more thermostable than that from tsr. In fact, the tsr(H258Y) gene served as a selectable marker for plasmid transformation of G. kaustophilus. This new marker could facilitate complex genetic modification of G. kaustophilus and potentially other Geobacillus spp.

  9. Progesterone inhibits proliferation and modulates expression of proliferation-Related genes in classical progesterone receptor-negative human BxPC3 pancreatic adenocarcinoma cells.

    Goncharov, Alexey I; Maslakova, Aitsana A; Polikarpova, Anna V; Bulanova, Elena A; Guseva, Alexandra A; Morozov, Ivan A; Rubtsov, Petr M; Smirnova, Olga V; Shchelkunova, Tatiana A


    Recent studies suggest that progesterone may possess anti-tumorigenic properties. However, a growth-modulatory role of progestins in human cancer cells remains obscure. With the discovery of a new class of membrane progesterone receptors (mPRs) belonging to the progestin and adipoQ receptor gene family, it becomes important to study the effect of this hormone on proliferation of tumor cells that do not express classical nuclear progesterone receptors (nPRs). To identify a cell line expressing high levels of mPRs and lacking nPRs, we examined mRNA levels of nPRs and three forms of mPRs in sixteen human tumor cell lines of different origin. High expression of mPR mRNA has been found in pancreatic adenocarcinoma BxPC3 cells, while nPR mRNA has not been detected in these cells. Western blot analysis confirmed these findings at the protein level. We revealed specific binding of labeled progesterone in these cells with affinity constant similar to that of human mPR expressed in yeast cells. Progesterone at high concentration of 20 μM significantly reduced the mRNA levels of proliferation markers Ki67 and PCNA, as well as of cyclin D1, and increased the mRNA levels of cyclin dependent kinase inhibitors p21 and p27. Progesterone (1 μM and 20 μM) significantly inhibited proliferative activity of BxPC3 cells. These results point to anti-proliferative effects of the progesterone high concentrations on BxPC3 cells and suggest that activation of mPRs may mediate this action. Our data are a starting point for further investigations regarding the application of progesterone in pancreatic cancer.

  10. Immunoexpression of angiogenesis and proliferation markers in patients treated with cyclosporin A.

    Afonso, M; Perrotti, V; Rapani, M; Iaculli, F; Piccirilli, M; Onuma, T; Shibli, J A; De Oliveira Bello, V; Sposto, M R; Artese, L


    In the present immunohistochemical study, the expression of vascular endothelial growth factor, nitric oxide synthase 1 and 3, and Ki-67 in the gingival tissues of renal transplant patients treated with cyclosporin A was assessed. Gingival overgrowth (GO) frequently occurs in transplant patients receiving immunosuppressive drugs such as cyclosporine and this gingival inflammation might play an important role in the pathogenesis of drug-induced GO. Twenty-eight human gingival biopsies were taken from healthy patients with chronic periodontitis (N.=14 control group), and from renal transplant recipients treated with cyclosporin A (N.=14 test group). The retrieved specimens were immunohistochemically processed and stained for vascular endothelial growth factor, nitric oxide synthase 1 and 3, and Ki-67. The levels of vascular endothelial growth factor, nitric oxide synthase 1 and 3, and Ki-67 were found to be significantly different among groups (P>0.001), with patients treated with cyclosporin A showing higher levels of all the analyzed markers compared to control group. In summary, the data from this pilot study suggests that the investigated factors have a role in the inflammation processes associated to immunosuppressive therapy. However, further studies with a larger sample population need to be conducted for an exhaustive knowledge of the mechanisms leading to GO.

  11. The impact of bisphosphonates on the osteoblast proliferation and Collagen gene expression in vitro

    Ziebart Thomas


    Full Text Available Abstract Background Bisphosphonates are widely used in the clinical treatment of bone diseases with increased bone resorption. In terms of side effects, they are known to be associated with osteonecrosis of the jaw (BONJ. The objective of this study was to evaluate the effect of bisphosphonates on osteoblast proliferation by cell count and gene expression analysis of cyclin D1 in vitro. Furthermore, the gene expression of the extracellular matrix protein collagen type I was evaluated. Nitrogen-containing and non-nitrogen-containing bisphosphonates have been compared on gene expression levels. Methods Human osteoblast obtained from hip bone were stimulated with zoledronate, ibandronate and clodronate at concentrations of 5 × 10-5M over the experimental periods of 1, 2, 5, 10 and 14 days. At each point in time, the cells were dissolved, the mRNA extracted, and the gene expression level of cyclin D1 and collagen type I were quantified by Real-Time RT-PCR. The gene expression was compared to an unstimulated osteoblast cell culture for control. Results The proliferation appeared to have been influenced only to a small degree by bisphosphonates. Zolendronate led to a lower cyclin D1 gene expression after 10 days. The collagen gene expression was enhanced by nitrogen containing bisphosphonates, decreased however after day 10. The non-nitrogen-containing bisphosphonate clodronate, however, did not significantly influence cyclin D1 and collagen gene expression. Conclusions The above data suggest a limited influence of bisphosphonates on osteoblast proliferation, except for zoledronate. The extracellular matrix production seems to be initially advanced and inhibited after 10 days. Interestingly, clodronate has little influence on osteoblast proliferation and extracellular matrix production in terms of cyclin D1 and collagen gene expression.

  12. Cre/lox-mediated marker gene excision in transgenic maize (Zea mays L.) plants.

    Zhang, W; Subbarao, S; Addae, P; Shen, A; Armstrong, C; Peschke, V; Gilbertson, L


    After the initial transformation and tissue culture process is complete, selectable marker genes, which are used in virtually all transformation approaches, are not required for the expression of the gene of interest in the transgenic plants. There are several advantages to removing the selectable marker gene after it is no longer needed, such as enabling the reuse of selectable markers and simplifying transgene arrays. We have tested the Cre/ lox system from bacteriophage P1 for its ability to precisely excise stably integrated marker genes from chromosomes in transgenic maize plants. Two strategies, crossing and autoexcision, have been tested and demonstrated. In the crossing strategy, plants expressing the Cre recombinase are crossed with plants bearing a transgene construct in which the selectable marker gene is flanked by directly repeated lox sites. Unlike previous reports in which incomplete somatic and germline excision were common, in our experiments complete somatic and germline marker gene excision occurred in the F(1) plants from most crosses with multiple independent Cre and lox lines. In the autoexcision strategy, the cre gene, under the control of a heat shock-inducible promoter, is excised along with the nptII marker gene. Our results show that a transient heat shock treatment of primary transgenic callus is sufficient for inducing cre and excising the cre and nptII genes. Genetic segregation and molecular analysis confirmed that marker gene removal is precise, complete and stable. The autoexcision strategy provides a way of removing the selectable marker gene from callus or other tissues such as embryos and kernels.

  13. Unbiased transcriptome signature of in vivo cell proliferation reveals pro- and antiproliferative gene networks.

    Cohen, Meital; Vecsler, Manuela; Liberzon, Arthur; Noach, Meirav; Zlotorynski, Eitan; Tzur, Amit


    Different types of mature B-cell lymphocytes are overall highly similar. Nevertheless, some B cells proliferate intensively, while others rarely do. Here, we demonstrate that a simple binary classification of gene expression in proliferating vs. resting B cells can identify, with remarkable selectivity, global in vivo regulators of the mammalian cell cycle, many of which are also post-translationally regulated by the APC/C E3 ligase. Consequently, we discover a novel regulatory network between the APC/C and the E2F transcription factors and discuss its potential impact on the G1-S transition of the cell cycle. In addition, by focusing on genes whose expression inversely correlates with proliferation, we demonstrate the inherent ability of our approach to also identify in vivo regulators of cell differentiation, cell survival, and other antiproliferative processes. Relying on data sets of wt, non-transgenic animals, our approach can be applied to other cell lineages and human data sets.

  14. HER4 selectively coregulates estrogen stimulated genes associated with breast tumor cell proliferation

    Han, Wen; Jones, Frank E., E-mail:


    Highlights: •HER4/4ICD is an obligate coactivator for 37% of estrogen regulated genes. •HER4/4ICD coactivated genes selectively regulate estrogen stimulated proliferation. •Estrogen stimulated tumor cell migration occurs independent of HER4/4ICD. •Disrupting HER4/4ICD and ER coactivated gene expression may suppress breast cancer. -- Abstract: The EGFR-family member HER4 undergoes regulated intramembrane proteolysis (RIP) to generate an intracellular domain (4ICD) that functions as a transcriptional coactivator. Accordingly, 4ICD coactivates the estrogen receptor (ER) and associates with ER at target gene promoters in breast tumor cells. However, the extent of 4ICD coactivation of ER and the functional significance of the 4ICD/ER transcriptional complex is unclear. To identify 4ICD coactivated genes we performed a microarray gene expression analysis of β-estradiol treated cells comparing control MCF-7 breast cancer cells to MCF-7 cells where HER4 expression was stably suppressed using a shRNA. In the MCF-7 cell line, β-estradiol significantly stimulated or repressed by 2-fold or more 726 or 53 genes, respectively. Significantly, HER4/4ICD was an obligate coactivator for 277 or 38% of the β-estradiol stimulated genes. Ingenuity Pathway Analysis of β-estradiol regulated genes identified significant associations with multiple cellular functions regulating cellular growth and proliferation, cell cycle progression, cancer metastasis, decreased hypoplasia, tumor cell migration, apoptotic resistance of tumor cells, and increased transcription. Genes coactivated by 4ICD displayed functional specificity by only significantly contributing to cellular growth and proliferation, cell cycle progression, and decreased hypoplasia. In direct concordance with these in situ results we show that HER4 knockdown in MCF-7 cells results in a loss of estrogen stimulated tumor cell proliferation and cell cycle progression, whereas, estrogen stimulated tumor cell migration was

  15. Molecular markers linked to the apple scab resistance gene Vbj derived from Malus baccata jackii.

    Gygax, M; Gianfranceschi, L; Liebhard, R; Kellerhals, M; Gessler, C; Patocchi, A


    Breeding for scab-resistant apple cultivars by pyramiding several resistance genes in the same genetic background is a promising way to control apple scab caused by the fungus Venturia inaequalis. To achieve this goal, DNA markers linked to the genes of interest are required in order to select seedlings with the desired resistance allele combinations. For several apple scab resistance genes, molecular markers are already available; but until now, none existed for the apple scab resistance gene Vbj originating from the crab apple Malus baccata jackii. Using bulk segregant analysis, three RAPD markers linked to Vbj were first identified. These markers were transformed into more reliable sequence-characterised amplified region (SCAR) markers that proved to be co-dominant. In addition, three SSR markers and one SCAR were identified by comparing homologous linkage groups of existing genetic maps. Discarding plants showing genotype-phenotype incongruence (GPI plants) plants, a linkage map was calculated. Vbj mapped between the markers CH05e03 (SSR) and T6-SCAR, at 0.6 cM from CH05e03 and at 3.9 cM from T6-SCAR. Without the removal of the GPI plants, Vbj was placed 15 cM away from the closest markers. Problems and pitfalls due to GPI plants and the consequences for mapping the resistance gene accurately are discussed. Finally, the usefulness of co-dominant markers for pedigree analysis is also demonstrated.

  16. E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis

    Müller, H; Bracken, A P; Vernell, R;


    The retinoblastoma protein (pRB) and its two relatives, p107 and p130, regulate development and cell proliferation in part by inhibiting the activity of E2F-regulated promoters. We have used high-density oligonucleotide arrays to identify genes in which expression changed in response to activatio...

  17. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells.

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes.

  18. A Cre/loxP-mediated self-activating gene excision system to produce marker gene free transgenic soybean plants.

    Li, Zhongsen; Xing, Aiqiu; Moon, Bryan P; Burgoyne, Susan A; Guida, Anthony D; Liang, Huiling; Lee, Catharina; Caster, Cheryl S; Barton, Joanne E; Klein, Theodore M; Falco, Saverio C


    Marker-gene-free transgenic soybean plants were produced by isolating a developmentally regulated embryo-specific gene promoter, app1, from Arabidopsis and developing a self-activating gene excision system using the P1 bacteriophage Cre/loxP recombination system. To accomplish this, the Cre recombinase gene was placed under control of the app1 promoter and, together with a selectable marker gene (hygromycin phosphotransferase), were cloned between two loxP recombination sites. This entire sequence was then placed between a constitutive promoter and a coding region for either beta-glucuronidase (Gus) or glyphosate acetyltransferase (Gat). Gene excision would remove the entire sequence between the two loxP sites and bring the coding region to the constitutive promoter for expression. Using this system marker gene excision occurred in over 30% of the stable transgenic events as indicated by the activation of the gus reporter gene or the gat gene in separate experiments. Transgenic plants with 1 or 2 copies of a functional excision-activated gat transgene and without any marker gene were obtained in T0 or T1 generation. This demonstrates the feasibility of using developmentally controlled promoters to mediate marker excision in soybean.

  19. Common Marker Genes Identified from Various Sample Types for Systemic Lupus Erythematosus.

    Peng-Fei Bing

    Full Text Available Systemic lupus erythematosus (SLE is a complex auto-immune disease. Gene expression studies have been conducted to identify SLE-related genes in various types of samples. It is unknown whether there are common marker genes significant for SLE but independent of sample types, which may have potentials for follow-up translational research. The aim of this study is to identify common marker genes across various sample types for SLE.Based on four public microarray gene expression datasets for SLE covering three representative types of blood-born samples (monocyte; peripheral blood mononuclear cell, PBMC; whole blood, we utilized three statistics (fold-change, FC; t-test p value; false discovery rate adjusted p value to scrutinize genes simultaneously regulated with SLE across various sample types. For common marker genes, we conducted the Gene Ontology enrichment analysis and Protein-Protein Interaction analysis to gain insights into their functions.We identified 10 common marker genes associated with SLE (IFI6, IFI27, IFI44L, OAS1, OAS2, EIF2AK2, PLSCR1, STAT1, RNASE2, and GSTO1. Significant up-regulation of IFI6, IFI27, and IFI44L with SLE was observed in all the studied sample types, though the FC was most striking in monocyte, compared with PBMC and whole blood (8.82-251.66 vs. 3.73-74.05 vs. 1.19-1.87. Eight of the above 10 genes, except RNASE2 and GSTO1, interact with each other and with known SLE susceptibility genes, participate in immune response, RNA and protein catabolism, and cell death.Our data suggest that there exist common marker genes across various sample types for SLE. The 10 common marker genes, identified herein, deserve follow-up studies to dissert their potentials as diagnostic or therapeutic markers to predict SLE or treatment response.

  20. Reduced expression of a gene proliferation signature is associated with enhanced malignancy in colon cancer.

    Anjomshoaa, A; Lin, Y-H; Black, M A; McCall, J L; Humar, B; Song, S; Fukuzawa, R; Yoon, H-S; Holzmann, B; Friederichs, J; van Rij, A; Thompson-Fawcett, M; Reeve, A E


    The association between cell proliferation and the malignant potential of colon cancer is not well understood. Here, we evaluated this association using a colon-specific gene proliferation signature (GPS). The GPS was derived by combining gene expression data obtained from the analysis of a cancer cell line model and a published colon crypt profile. The GPS was overexpressed in both actively cycling cells in vitro and the proliferate compartment of colon crypts. K-means clustering was used to independantly stratify two cohorts of colon tumours into two groups with high and low GPS expression. Notably, we observed a significant association between reduced GPS expression and an increased likelihood of recurrence (P cancer malignancy and increased proliferation, by applying our GPS to public breast cancer data. In this study, we show that reduced proliferation is a biological feature characterizing the majority of aggressive colon cancers. This contrasts with many other carcinomas such as breast cancer. Investigating the reasons underlying this unusual observation may provide important insight into the biology of colon cancer progression and putative novel therapy options.

  1. Human gastric mucins differently regulate Helicobacter pylori proliferation, gene expression and interactions with host cells.

    Emma C Skoog

    Full Text Available Helicobacter pylori colonizes the mucus niche of the gastric mucosa and is a risk factor for gastritis, ulcers and cancer. The main components of the mucus layer are heavily glycosylated mucins, to which H. pylori can adhere. Mucin glycosylation differs between individuals and changes during disease. Here we have examined the H. pylori response to purified mucins from a range of tumor and normal human gastric tissue samples. Our results demonstrate that mucins from different individuals differ in how they modulate both proliferation and gene expression of H. pylori. The mucin effect on proliferation varied significantly between samples, and ranged from stimulatory to inhibitory, depending on the type of mucins and the ability of the mucins to bind to H. pylori. Tumor-derived mucins and mucins from the surface mucosa had potential to stimulate proliferation, while gland-derived mucins tended to inhibit proliferation and mucins from healthy uninfected individuals showed little effect. Artificial glycoconjugates containing H. pylori ligands also modulated H. pylori proliferation, albeit to a lesser degree than human mucins. Expression of genes important for the pathogenicity of H. pylori (babA, sabA, cagA, flaA and ureA appeared co-regulated in response to mucins. The addition of mucins to co-cultures of H. pylori and gastric epithelial cells protected the viability of the cells and modulated the cytokine production in a manner that differed between individuals, was partially dependent of adhesion of H. pylori to the gastric cells, but also revealed that other mucin factors in addition to adhesion are important for H. pylori-induced host signaling. The combined data reveal host-specific effects on proliferation, gene expression and virulence of H. pylori due to the gastric mucin environment, demonstrating a dynamic interplay between the bacterium and its host.

  2. Identiifcation of Human Hepatocyte Proliferation Related Gene C2orf69

    Ren-wen Zhang; Yong Qiao; Xiao-hua Hao; Hong-min Li; Hui Ren; Xiao-jing Zhang; Hong-shan Wei; Xiao-yuan Xu


    Objective To construct the prokaryotic expression vector pET-32a(+)-C2orf69 and induce the expression of recombinant proteins in vitro. Then the possible effects of recombinant protein on cell proliferation was observed and rabbit-anti-C2orf69 protein polyclonal antibodies was obtained. Methods Gene fragment of C2orf69 was ampliifed by PCR and then prokaryotic expression plasmid pET-32a(+)-C2orf69 was constructed. Recombinant protein C2orf69 expression was identiifed by SDS-PAGE and Western blot. The white-ear rabbits were immunized with purified recombinant protein C2orf69, and the potency and speciifcity of polyclonal antibody were evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blot. Also, different liver cells were incubated with recombinant protein C2orf69 in vitro. Results C2orf69 gene fragment was successfully ampliifed, results of gene sequencing were consistent with the sequence in GenBank. Recombinant protein of C2orf69 was successfully induced and expressed. The polyclonal antibody titer was up to 1︰1 280 000 through enzyme-linked immunosorbent assay. Results of cell proliferation showed that the recombinant protein could inhibit the proliferation of different liver cells. Conclusions The recombinant protein C2orf69 could inhibit the proliferation of different liver cells, and we speculated that it may be a widely roled inhibitor of hepatocyte proliferation. Our experiment showed that the proliferation inhibition of cells may be realized by G1 phase extending and S phase shortening.

  3. Development of Random Amplified Polymorphism DNA Markers Linked to Powdery Mildew Resistance Gene in Melon

    Budi Setiadi Daryono


    Full Text Available A random amplified polymorphic DNA (RAPD marker linked to powdery mildew resistance gene (Pm-I in melon PI 371795 was reported. However, the RAPD marker has problem in scoring. To detect powdery mildew resistance gene (Pm-I in melon accurately, the RAPD marker was cloned and sequenced to design sequence characterized amplified region (SCAR markers. SCAPMAR5 marker derived from pUBC411 primer yielded a single DNA band at 1061 bp. Segregation of SCAPMAR5 marker in bulk of F2 plants demonstrated that the marker was co-segregated with RAPD marker from which the SCAR marker was originated. Moreover, results of SCAR analysis in diverse melons showed SCAPMAR5 primers obtained a single 1061 bp linked to Pm-I in resistant melon PI 371795 and PMAR5. On the other hand, SCAPMAR5 failed to detect Pm-I in susceptible melons. Results of this study revealed that SCAR analysis not only confirmed melons that had been clearly scored for resistance to Pm-I evaluated by RAPD markers, but also clarified the ambiguous resistance results obtained by the RAPD markers.   Key words: Cucumis melo L., Pm-I, RAPD, SCAPMAR5

  4. Identification of peroxisome-proliferator responsive element in the mouse HSL gene.

    Yajima, Hiroaki; Kobayashi, Yumie; Kanaya, Tomoka; Horino, Yoko


    Hormone-sensitive lipase (HSL) catalyzes the rate-limiting step of lipolysis in adipose tissue. Several studies suggest that protein phosphorylation regulates the HSL enzymatic activity. On the other hand, the precise mechanism of the transcriptional regulation of the HSL gene remains to be elucidated. Here, we identified a functional peroxisome-proliferator responsive element (PPRE) in the mouse HSL promoter by reporter assay in CV-1 cells using serial deletion and point mutants of the 5'-flanking region. Chromatin immunoprecipitation (ChIP) analysis revealed that both peroxisome-proliferator activated receptor (PPARgamma) and retinoid X receptor (RXRalpha) interacted with the region. Binding of the PPARgamma/RXRalpha heterodimer to the PPRE sequence was also confirmed by electrophoretic mobility shift assay. These results indicate that the HSL gene is transcriptionally regulated by PPARgamma/RXRalpha heterodimer, and suggest that a cis-acting element regulates the HSL gene expression.

  5. Canine candidate genes for dilated cardiomyopathy: annotation of and polymorphic markers for 14 genes

    van Oost Bernard A


    Full Text Available Abstract Background Dilated cardiomyopathy is a myocardial disease occurring in humans and domestic animals and is characterized by dilatation of the left ventricle, reduced systolic function and increased sphericity of the left ventricle. Dilated cardiomyopathy has been observed in several, mostly large and giant, dog breeds, such as the Dobermann and the Great Dane. A number of genes have been identified, which are associated with dilated cardiomyopathy in the human, mouse and hamster. These genes mainly encode structural proteins of the cardiac myocyte. Results We present the annotation of, and marker development for, 14 of these genes of the dog genome, i.e. α-cardiac actin, caveolin 1, cysteine-rich protein 3, desmin, lamin A/C, LIM-domain binding factor 3, myosin heavy polypeptide 7, phospholamban, sarcoglycan δ, titin cap, α-tropomyosin, troponin I, troponin T and vinculin. A total of 33 Single Nucleotide Polymorphisms were identified for these canine genes and 11 polymorphic microsatellite repeats were developed. Conclusion The presented polymorphisms provide a tool to investigate the role of the corresponding genes in canine Dilated Cardiomyopathy by linkage analysis or association studies.

  6. Transcriptional and posttranscriptional regulation of the proliferating cell nuclear antigen gene.


    The steady-state mRNA levels of the proliferating cell nuclear antigen (PCNA) gene are growth regulated. In a previous paper (L. Ottavio, C.-D. Chang, M. G. Rizzo, S. Travali, C. Casadevall, and R. Baserga, Mol. Cell. Biol. 10:303-309, 1990), we reported that introns (especially intron 4) participate in growth regulation of the PCNA gene. We have now investigated the role of the 5'-flanking sequence of the human PCNA gene stably transfected into BALB/c 3T3 cells. Promoters of different length...

  7. Identification of glucocorticoid-regulated genes that control cell proliferation during murine respiratory development.

    Bird, Anthony D; Tan, Kheng H; Olsson, P Fredrik; Zieba, Malgorzata; Flecknoe, Sharon J; Liddicoat, Douglas R; Mollard, Richard; Hooper, Stuart B; Cole, Timothy J


    Glucocorticoids play a vital role in fetal respiratory development and act via the intracellular glucocorticoid receptor (GR) to regulate transcription of key target genes. GR-null mice die at birth due to respiratory dysfunction associated with hypercellularity and atelectasis. To identify events associated with this lung phenotype we examined perinatal cellular proliferation rates and apoptotic indices. We demonstrate that compared to wild-type controls, day 18.5 postcoitum (p.c.) GR-null mouse lungs display significantly increased cell proliferation rates (1.8-fold P < 0.05) and no change in apoptosis. To examine underlying molecular mechanisms, we compared whole genome expression profiles by microarray analysis at 18.5 days p.c. Pathways relating to cell proliferation, division and cell cycle were significantly down-regulated while pathways relating to carbohydrate metabolism, kinase activities and immune responses were significantly up-regulated. Differential levels of gene expression were verified by quantitative-RT-PCR and/or Northern analysis. Key regulators of proliferation differentially expressed in the lung of 18.5 p.c. GR-null lungs included p21 CIP1 (decreased 2.9-fold, P < 0.05), a negative regulator of the cell cycle, and Mdk (increased 6.0-fold, P < 0.05), a lung growth factor. The more under-expressed genes in 18.5 p.c. GR-null lungs included Chi3l3 (11-fold, P < 0.05), a macrophage inflammatory response gene and Ela1 (9.4-fold, P < 0.05), an extracellular matrix remodeling enzyme. Our results demonstrate that GR affects the transcriptional status of a number of regulatory processes during late fetal lung development. Amongst these processes is cell proliferation whereby GR induces expression of cell cycle repressors while suppressing induction of a well characterized cell cycle stimulator.

  8. Mesenchymal stromal cell proliferation, gene expression and protein production in human platelet-rich plasma-supplemented media.

    Amable, Paola Romina; Teixeira, Marcus Vinicius Telles; Carias, Rosana Bizon Vieira; Granjeiro, José Mauro; Borojevic, Radovan


    Platelet-rich plasma (PRP) is increasingly used as a cell culture supplement, in order to reduce the contact of human cells with animal-derived products during in vitro expansion. The effect of supplementation changes on cell growth and protein production is not fully characterized. Human mesenchymal stromal cells from bone marrow, adipose tissue and Wharton's Jelly were isolated and cultured in PRP-supplemented media. Proliferation, in vitro differentiation, expression of cell surface markers, mRNA expression of key genes and protein secretion were quantified. 10% PRP sustained five to tenfold increased cell proliferation as compared to 10% fetal bovine serum. Regarding cell differentiation, PRP reduced adipogenic differentiation and increased calcium deposits in bone marrow and adipose tissue-mesenchymal stromal cells. Wharton's Jelly derived mesenchymal stromal cells secreted higher concentrations of chemokines and growth factors than other mesenchymal stromal cells when cultured in PRP-supplemented media. Bone marrow derived mesenchymal stromal cells secreted higher concentrations of pro-inflammatory and pro-angiogenic proteins. Mesenchymal stromal cells isolated from adipose tissue secreted higher amounts of extracellular matrix components. Mesenchymal stromal cells purified from different tissues have distinct properties regarding differentiation, angiogenic, inflammatory and matrix remodeling potential when cultured in PRP supplemented media. These abilities should be further characterized in order to choose the best protocols for their therapeutic use.

  9. Low frequency pulsed electromagnetic field affects proliferation, tissue-specific gene expression, and cytokines release of human tendon cells.

    de Girolamo, L; Stanco, D; Galliera, E; Viganò, M; Colombini, A; Setti, S; Vianello, E; Corsi Romanelli, M M; Sansone, V


    Low frequency pulsed electromagnetic field (PEMF) has proven to be effective in the modulation of bone and cartilage tissue functional responsiveness, but its effect on tendon tissue and tendon cells (TCs) is still underinvestigated. PEMF treatment (1.5 mT, 75 Hz) was assessed on primary TCs, harvested from semitendinosus and gracilis tendons of eight patients, under different experimental conditions (4, 8, 12 h). Quantitative PCR analyses were conducted to identify the possible effect of PEMF on tendon-specific gene transcription (scleraxis, SCX and type I collagen, COL1A1); the release of pro- and anti-inflammatory cytokines and of vascular endothelial growth factor (VEGF) was also assessed. Our findings show that PEMF exposure is not cytotoxic and is able to stimulate TCs' proliferation. The increase of SCX and COL1A1 in PEMF-treated cells was positively correlated to the treatment length. The release of anti-inflammatory cytokines in TCs treated with PEMF for 8 and 12 h was significantly higher in comparison with untreated cells, while the production of pro-inflammatory cytokines was not affected. A dramatically higher increase of VEGF-A mRNA transcription and of its related protein was observed after PEMF exposure. Our data demonstrated that PEMF positively influence, in a dose-dependent manner, the proliferation, tendon-specific marker expression, and release of anti-inflammatory cytokines and angiogenic factor in a healthy human TCs culture model.

  10. Role of peroxisome proliferator-activated receptors gene polymorphisms in type 2 diabetes and metabolic syndrome.

    Dong, Chen; Zhou, Hui; Shen, Chong; Yu, Lu-Gang; Ding, Yi; Zhang, Yong-Hong; Guo, Zhi-Rong


    Metabolic syndrome (MetS) and type 2 diabetes mellitus (T2DM) are the serious public health problems worldwide. Moreover, it is estimated that MetS patients have about five-fold greater risk of the T2DM development compared with people without the syndrome. Peroxisome proliferator-activated receptors are a subgroup of the nuclear hormone receptor superfamily of ligand-activated transcription factors which play an important role in the pathogenesis of MetS and T2DM. All three members of the peroxisome proliferator-activated receptor (PPAR) nuclear receptor subfamily, PPARα, PPARβ/δ and PPARγ are critical in regulating insulin sensitivity, adipogenesis, lipid metabolism, and blood pressure. Recently, more and more studies indicated that the gene polymorphism of PPARs, such as Leu(162)Val and Val(227)Ala of PPARα, +294T > C of PPARβ/δ, Pro(12)Ala and C1431T of PPARγ, are significantly associated with the onset and progressing of MetS and T2DM in different population worldwide. Furthermore, a large body of evidence demonstrated that the glucose metabolism and lipid metabolism were influenced by gene-gene interaction among PPARs genes. However, given the complexity pathogenesis of metabolic disease, it is unlikely that genetic variation of a single locus would provide an adequate explanation of inter-individual differences which results in diverse clinical syndromes. Thus, gene-gene interactions and gene-environment interactions associated with T2DM and MetS need future comprehensive studies.

  11. The exceptional stem cell system of Macrostomum lignano: Screening for gene expression and studying cell proliferation by hydroxyurea treatment and irradiation

    Eichberger Paul


    Full Text Available Abstract Background Flatworms are characterized by an outstanding stem cell system. These stem cells (neoblasts can give rise to all cell types including germ cells and power the exceptional regenerative capacity of many flatworm species. Macrostomum lignano is an emerging model system to study stem cell biology of flatworms. It is complementary to the well-studied planarians because of its small size, transparency, simple culture maintenance, the basal taxonomic position and its less derived embryogenesis that is more closely related to spiralians. The development of cell-, tissue- and organ specific markers is necessary to further characterize the differentiation potential of flatworm stem cells. Large scale in situ hybridization is a suitable tool to identify possible markers. Distinguished genes identified in a large scale screen in combination with manipulation of neoblasts by hydroxyurea or irradiation will advance our understanding of differentiation and regulation of the flatworm stem cell system. Results We have set up a protocol for high throughput large scale whole mount in situ hybridization for the flatworm Macrostomum lignano. In the pilot screen, a number of cell-, tissue- or organ specific expression patterns were identified. We have selected two stem cell- and germ cell related genes – macvasa and macpiwi – and studied effects of hydroxyurea (HU treatment or irradiation on gene expression. In addition, we have followed cell proliferation using a mitosis marker and bromodeoxyuridine labeling of S-phase cells after various periods of HU exposure or different irradiation levels. HU mediated depletion of cell proliferation and HU induced reduction of gene expression was used to generate a cDNA library by suppressive subtractive hybridization. 147 differentially expressed genes were sequenced and assigned to different categories. Conclusion We show that Macrostomum lignano is a suitable organism to perform high throughput large

  12. Molecular markers of nuclear restoration gene Rf1 in sunflower using bulked segregant analysis-RAPD

    季静; 王罡; E.Belhassen; H.Serieys; A.Berville


    Restoration of cytoplasmic male sterility (CMS) in sunflower was demonstrated to be controlled by polygenes by analysing 982 effective crosses among 109 self-crossed lines and 16 CMS lines. Two self-crossed lines and one CMS line with distinct genotypes were applied to creation of segregating populations for DNA bulks of the target gene Rfl. Bulked DNA was prepared in order to investigate single gene Rfl and its gene marker among polygenic characters at the same genetic background. Using 80 10-mer operon primers, 620 RAPD reactions were carried out between fertile and sterile DNA bulks. In about 800 loci, primary results showed that 8 were related to the restoration genes. Furthermore. 2 were confirmed as RAPD markers for gene Rfl by examining 9 maintenance and 7 restoration lines. This method is the improvement for bulked segregant analysis[1] with which markers of single gene of target can be identified rapidly among polygenic characters.

  13. Twist1 directly regulates genes that promote cell proliferation and migration in developing heart valves.

    Mary P Lee

    Full Text Available Twist1, a basic helix-loop-helix transcription factor, is expressed in mesenchymal precursor populations during embryogenesis and in metastatic cancer cells. In the developing heart, Twist1 is highly expressed in endocardial cushion (ECC valve mesenchymal cells and is down regulated during valve differentiation and remodeling. Previous studies demonstrated that Twist1 promotes cell proliferation, migration, and expression of primitive extracellular matrix (ECM molecules in ECC mesenchymal cells. Furthermore, Twist1 expression is induced in human pediatric and adult diseased heart valves. However, the Twist1 downstream target genes that mediate increased cell proliferation and migration during early heart valve development remain largely unknown. Candidate gene and global gene profiling approaches were used to identify transcriptional targets of Twist1 during heart valve development. Candidate target genes were analyzed for evolutionarily conserved regions (ECRs containing E-box consensus sequences that are potential Twist1 binding sites. ECRs containing conserved E-box sequences were identified for Twist1 responsive genes Tbx20, Cdh11, Sema3C, Rab39b, and Gadd45a. Twist1 binding to these sequences in vivo was determined by chromatin immunoprecipitation (ChIP assays, and binding was detected in ECCs but not late stage remodeling valves. In addition identified Twist1 target genes are highly expressed in ECCs and have reduced expression during heart valve remodeling in vivo, which is consistent with the expression pattern of Twist1. Together these analyses identify multiple new genes involved in cell proliferation and migration that are differentially expressed in the developing heart valves, are responsive to Twist1 transcriptional function, and contain Twist1-responsive regulatory sequences.

  14. Interleukin-2 expression and glioma cell proliferation following Vaceinia vector gene transfection in vivo

    Xiaogang Wang; Xuezhong Wei; Jiangqiu Liu


    BACKGROUND: The effectiveness of gene therapy is closely related to the efficiency of vector transfection and expression.OBJECTIVE: This study was designed to transfect a human brain glioma cell line with recombinant Vaccinia virus expressing the interleukin-2 (rVV-IL-2) gene, and to observe IL-2 expression and glioma cell proliferation potential after transfection. DESIGN: Experimental observation. SETTING: Department of Neurosurgery, Shenyang Military Area Command of Chinese PLA. MATERIALS: The rVV-IL-2 vectors were obtained through homologous recombination and screening in the Second Military Medical University of Chinese PLA. The human brain glioma cell line and IL-2-dependent cells were produced by the Second Military Medical University of Chinese PLA. Human IL-2 was produced by Genzyme Corporation. MAIN OUTCOME MEASURES: IL-2 expression at different time points after transfection of human brain glioma cells with varying MOI of Vaccinia viral vectors; in vitro proliferation capacity of human brain glioma cells among the 4 groups. RESULTS: IL-2 expression was detectable 4 hours after Vaccinia viral vector transfection and reached 300 kU/L by 8 hours. There was no significant difference in the proliferating rate of human brain glioma cells among the 4 groups (P > 0.05).CONCLUSION: Vaccinia viral vectors can transfect human brain glioma cells in vitro and express high levels of IL-2. Vaccinia virus and high IL-2 expression do not influence the proliferation rate of human brain glioma cells in vitro.

  15. Cadmium induces Wnt signaling to upregulate proliferation and survival genes in sub-confluent kidney proximal tubule cells

    Wolff Natascha A


    Full Text Available Abstract Background The class 1 carcinogen cadmium (Cd2+ disrupts the E-cadherin/β-catenin complex of epithelial adherens junctions (AJs and causes renal cancer. Deregulation of E-cadherin adhesion and changes in Wnt/β-catenin signaling are known to contribute to carcinogenesis. Results We investigated Wnt signaling after Cd2+-induced E-cadherin disruption in sub-confluent cultured kidney proximal tubule cells (PTC. Cd2+ (25 μM, 3-9 h caused nuclear translocation of β-catenin and triggered a Wnt response measured by TOPflash reporter assays. Cd2+ reduced the interaction of β-catenin with AJ components (E-cadherin, α-catenin and increased binding to the transcription factor TCF4 of the Wnt pathway, which was upregulated and translocated to the nucleus. While Wnt target genes (c-Myc, cyclin D1 and ABCB1 were up-regulated by Cd2+, electromobility shift assays showed increased TCF4 binding to cyclin D1 and ABCB1 promoter sequences with Cd2+. Overexpression of wild-type and mutant TCF4 confirmed Cd2+-induced Wnt signaling. Wnt signaling elicited by Cd2+ was not observed in confluent non-proliferating cells, which showed increased E-cadherin expression. Overexpression of E-cadherin reduced Wnt signaling, PTC proliferation and Cd2+ toxicity. Cd2+ also induced reactive oxygen species dependent expression of the pro-apoptotic ER stress marker and Wnt suppressor CHOP/GADD153 which, however, did not abolish Wnt response and cell viability. Conclusions Cd2+ induces Wnt signaling in PTC. Hence, Cd2+ may facilitate carcinogenesis of PTC by promoting Wnt pathway-mediated proliferation and survival of pre-neoplastic cells.

  16. [Effects of Sam68 gene silence on proliferation of acute T lymphoblastic leukemia cell line Jurkat].

    Wang, Chi-Juan; Xu, Hua; Zhang, Hai-Rui; Wang, Jian; Lin, Ya-Ni; Pang, Tian-Xiang; Li, Qing-Hua


    This study was purpose to investigate the effect of Sam68 gene silence on proliferation of human acute T lymphoblastic leukemia cell line Jurkat. The sequence of shRNA targeting the site 531-552 of Sam68 mRNA was designed and chemically synthesized, then a single-vector lentiviral, Tet-inducible shRNA-Sam68 system (pLKO-Tet-On) was constructed; next the Jurkat cells were infected with lentivirus to create stable cell clones with regulatable Sam68 gene expression. The inhibitory efficiency of Sam68 gene was assayed by Real-time PCR and Western blot; the cell activity of Jurkat cells was detected with MTT assay; the change of colony forming potential of Jurkat cells was analyzed by colony forming test; the cell cycle distribution was tested by flow cytometry. The results indicated that the expression of Sam68 in experimental cells was statistically decreased as compared with that of the control cells; the cells activity and colony forming capacity of the Jurkat cells with Sam68 gene silence were significantly inhibited; with Sam68 gene silencing, the percentage of S phase cells was significantly increased, while the percentage of G2 phase cells was significantly decreased. It is concluded that the silencing Sam68 gene using shRNA interference can effectively inhibit the proliferation of human acute T lymphoblastic leukemia cell line Jurkat.

  17. Identification of putative candidate gene markers for grain zinc ...



    Jan 29, 2014 ... with 96 rice genotypes showed three markers (OsZIP8a, OsNAC and OsZIP4b) with phenotypic variation of 11.0, 5.8 .... tent in wheat mapping population, ranging from 19.9 to .... Genotype×environment interaction for iron con-.

  18. Identification of Molecular Markers Linked to TuMV Resistant Gene in Cabbage

    GAO Jinping; WANG Chao; LIU Ying


    A total of 144 F2 individuals were obtained from the crossing between 1047 (susceptible) and A21 (resistant).Two RAPD markers were screened out in 200 random primers using BSA(Bulked Segregant Analysis). Two RAPD markers, designated as AG13/2000 and U16/660,were 7.7 cM and 8.38 cM apart from the TuMV resistant gene,respectively. The two RAPD fragments were converted to SCAR markers.SCAR markers were confined in germplasm.

  19. Suicide Vector Construction of Haemophilus parasuis hhd B Gene Marker-free Deleted

    Song Shuai; Li Miao; Li Yan; Jiang Zhiyong; Cai Rujian; Yang Dongxia; Li Chunling


    To construct the suicide vector of hhd B gene marker-free mutant in Haemophilus parasuis( HPS),two pairs of specific primers were designed and synthesized according to the hhd B gene upstream and downstream sequences of HPS published in Gen Bank. The hhd B gene upstream and downstream sequences were amplified by PCR,which were further ligated( hhd B-up + down) through overlapping PCR method. NotⅠand SalⅠrestriction enzyme sites were introduced on both ends of the ligated sequence. After the corresponding digestion,the hhd B-up + down sequence was directionally cloned to the suicide plasmid vector p EMOC2. Results showed that the suicide vector of hhd B gene marker-free deleted( p EMOC2Δhhd B) with stable inheritance in E. coli β2155 strain was successfully obtained,thereby laying the foundation for construction of HPS-hhd B gene marker-free mutant strain.

  20. Candidate gene markers for sperm quality and fertility in bulls

    Chinmoy Mishra


    Full Text Available Fertility is one of the primary traits of reproduction in bulls. Decrease in fertility is a multifactorial condition and is verydifficult to diagnose. Among various causes genetic abnormality holds a major share. By identifying various genes that haveeffects on fertility the genetic cause behind subferility can be explored and also other non genetic factors can be identified.Advancement of molecular genetic tools now easily enables us to explore individual genes in animals. Identification of thesegenes will eventually lead to genome assembly and development of novel tools for analysing complex genetic traits. Thispaper gives a brief idea about the candidate genes for bull fertility, including genes encoding hormones and their receptors,proteins of the seminal plasma, proteins involved in spermatozoa-ovum binding and genes influencing sexual development.The chromosomal location and gene structure are described, based on the bovine genome assembly.

  1. A new monoclonal antibody against DNA ligase I is a suitable marker of cell proliferation in cultured cell and tissue section samples

    B Vitolo


    Full Text Available The extensive characterization of the replicative human DNA ligase I (LigI undertaken in the last decade demonstrated that the level of this protein strongly correlates with the rate of cell proliferation. This may allow to expand the repertoire of clinical biomarkers for the analysis of cell proliferation.We have produced a new monoclonal antibody (5H5 against LigI and exploited it as cell proliferation marker in Western blotting and immunofluorescence as well as in immunohistochemistry on paraffin tissue sections. The Western blot analysis showed that the LigI level detected by 5H5 antibody is high in all proliferating cells. On the contrary the protein is down regulated in resting human fibroblast and peripheral blood lymphocytes. Immunofluorescence analysis on cultured HeLa cells showed that 5H5 antibody labels all proliferating cells and displays the same staining pattern of BrdU in S-phase nuclei. Finally the analysis of serial sections of inflamed tonsils and NHL lymph nodes (either frozen or paraffin embedded demonstrated that 5H5 marks the same population of cells as the Ki-67 antibody. Our results demonstrate that 5H5 antibody is a valuable tool for labeling proliferating cells that can be conveniently used in Western blotting, immunocytochemistry and immunohistochemistry.


    GU Feng; ZHANG Qing-li; GUO Xiao-chun; LI Tao; WANG Hong-gang


    Powdery mildew is one of the most serious diseases of wheat in China. In this paper,bulked segregant analysis (BSA) was used to search for randomly amplified polymorphic DNA (RAPD) markers linked to the Pm12 gene,which confers resistance to the powdery mildew in wheat. 200 decamer primers were screened and one RAPD marker (S1071900) was identified to be linked to Pm 12 in coupling phase, and their genetic distance is 11.98 ±4.00cM. This marker can be used for marker - assisted selection in wheat breeding for the identification or pyramiding of Pm12 with other resistance genes.Key Words: Wheat, RAPD, Powdery mildew, resistance genes.

  3. Development of genomic SSR markers for fingerprinting lettuce (Lactuca sativa L.) cultivars and mapping genes.

    Rauscher, Gilda; Simko, Ivan


    Lettuce (Lactuca sativa L.) is the major crop from the group of leafy vegetables. Several types of molecular markers were developed that are effectively used in lettuce breeding and genetic studies. However only a very limited number of microsattelite-based markers are publicly available. We have employed the method of enriched microsatellite libraries to develop 97 genomic SSR markers. Testing of newly developed markers on a set of 36 Lactuca accession (33 L. sativa, and one of each L. serriola L., L. saligna L., and L. virosa L.) revealed that both the genetic heterozygosity (UHe = 0.56) and the number of loci per SSR (Na = 5.50) are significantly higher for genomic SSR markers than for previously developed EST-based SSR markers (UHe = 0.32, Na = 3.56). Fifty-four genomic SSR markers were placed on the molecular linkage map of lettuce. Distribution of markers in the genome appeared to be random, with the exception of possible cluster on linkage group 6. Any combination of 32 genomic SSRs was able to distinguish genotypes of all 36 accessions. Fourteen of newly developed SSR markers originate from fragments with high sequence similarity to resistance gene candidates (RGCs) and RGC pseudogenes. Analysis of molecular variance (AMOVA) of L. sativa accessions showed that approximately 3% of genetic diversity was within accessions, 79% among accessions, and 18% among horticultural types. The newly developed genomic SSR markers were added to the pool of previously developed EST-SSRs markers. These two types of SSR-based markers provide useful tools for lettuce cultivar fingerprinting, development of integrated molecular linkage maps, and mapping of genes.

  4. Genome polymorphism markers and stress genes expression for ...



    Jun 11, 2014 ... environmental stress through investigating SOD and PAL gene expression and also the genetic relationship .... obtained from the gene bank ( under accession number ... stored at -20°C for further work. Primers ...

  5. Musashi1 modulates cell proliferation genes in the medulloblastoma cell line Daoy

    Hung Jaclyn Y


    Full Text Available Abstract Background Musashi1 (Msi1 is an RNA binding protein with a central role during nervous system development and stem cell maintenance. High levels of Msi1 have been reported in several malignancies including brain tumors thereby associating Msi1 and cancer. Methods We used the human medulloblastoma cell line Daoy as model system in this study to knock down the expression of Msi1 and determine the effects upon soft agar growth and neurophere formation. Quantitative RT-PCR was conducted to evaluate the expression of cell proliferation, differentiation and survival genes in Msi1 depleted Daoy cells. Results We observed that MSI1 expression was elevated in Daoy cells cultured as neurospheres compared to those grown as monolayer. These data indicated that Msi1 might be involved in regulating proliferation in cancer cells. Here we show that shRNA mediated Msi1 depletion in Daoy cells notably impaired their ability to form colonies in soft agar and to grow as neurospheres in culture. Moreover, differential expression of a group of Notch, Hedgehog and Wnt pathway related genes including MYCN, FOS, NOTCH2, SMO, CDKN1A, CCND2, CCND1, and DKK1, was also found in the Msi1 knockdown, demonstrating that Msi1 modulated the expression of a subset of cell proliferation, differentiation and survival genes in Daoy. Conclusion Our data suggested that Msi1 may promote cancer cell proliferation and survival as its loss seems to have a detrimental effect in the maintenance of medulloblastoma cancer cells. In this regard, Msi1 might be a positive regulator of tumor progression and a potential target for therapy.

  6. Mutant acetolactate synthase (ALS) gene as a selectable marker for Agrobacterium-mediated transformation of soybean

    Chen Shiyun; Zhang Yong


    Soybean is one of the crops most difficult to be manipulated in vitro. Although several soybean transformation systems with different selectable marker genes have been reported, e.g. antibiotic (kanamycin or hygromycin) resistant genes and herbicide ( glufosinate, glyphosate) resistant selectable marker genes, all the selectable markers used were from bacteria origin. To find suitable selectable marker gene from plant origin for soybean transformation, a mutant acetolactate synthase (ALS) gene from Arabidopsis thaliana was tested for Agrobacterium-mediated soybean embryo axis transformation with the herbicide Arsenal as the selective agent. Transgenic soybean plants were obtained after the herbicide selection and the To transgenic lines showed resistance to the herbicide at a concentration of 100 g/ha. ALS enzyme assay of To transgenic line also showed higher activity compared to the wild type control plant.PCR analysis of the T1 transgenic lines confirmed the integration and segregation of the transgene. Taken together, our results showed that the mutant ALS gene is a suitable selectable marker for soybean transformation.

  7. Predicting survival outcomes using subsets of significant genes in prognostic marker studies with microarrays

    Matsui Shigeyuki


    Full Text Available Abstract Background Genetic markers hold great promise for refining our ability to establish precise prognostic prediction for diseases. The development of comprehensive gene expression microarray technology has allowed the selection of relevant marker genes from a large pool of candidate genes in early-phased, developmental prognostic marker studies. The primary analytical task in such studies is to select a small fraction of relevant genes, typically from a list of significant genes, for further investigation in subsequent studies. Results We develop a methodology for predicting survival outcomes using subsets of significant genes in prognostic marker studies with microarrays. Key components in this methodology include building prediction models, assessing predictive performance of prediction models, and assessing significance of prediction results. As particular specifications, we assume Cox proportional hazard models with a compound covariate. For assessing predictive accuracy, we propose to use the cross-validated log partial likelihood. To assess significance of prediction results, we apply permutation procedures in cross-validated prediction. As an additional key component peculiar to prognostic prediction, we also consider incorporation of standard prognostic factors. The methodology is evaluated using both simulated and real data. Conclusion The developed methodology for prognostic prediction using a subset of significant genes can provide new insights based on predictive capability, possibly incorporating standard prognostic factors, in selecting a fraction of relevant genes for subsequent studies.

  8. Use of the alr gene as a food-grade selection marker in lactic acid bacteria

    Bron, P.A.; Benchimol, M.G.; Lambert, J.; Palumbo, E.; Deghorain, M.; Delcour, J.; Vos, de W.M.; Kleerebezem, M.; Hols, P.


    Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC, which catalyzes the interconversion of D-alanine and L-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection marker

  9. IL-27 inhibits lymphatic endothelial cell proliferation by STAT1-regulated gene expression

    Nielsen, Sebastian Rune; Hammer, Troels; Gibson, Josefine


    OBJECTIVE: IL-27 belongs to the IL-12 family of cytokines and is recognized for its role in Th cell differentiation and as an inhibitor of tumor-angiogenesis. The purpose of this study was to investigate the effect of IL-27 on proliferation of lymphatic endothelial cells to gain insight into the ......OBJECTIVE: IL-27 belongs to the IL-12 family of cytokines and is recognized for its role in Th cell differentiation and as an inhibitor of tumor-angiogenesis. The purpose of this study was to investigate the effect of IL-27 on proliferation of lymphatic endothelial cells to gain insight......U kits and the role of STAT1 and chemokines was determined by use of siRNA and recombinant proteins. RESULTS: Stimulation of lymphatic endothelial cell cultures with IL-27 induced JAK dependent phosphorylation of STAT1 and STAT3 and inhibited lymphatic endothelial cell proliferation and migration....... Expression of CXCL10 and CXCL11, both STAT1 target genes, were profoundly up-regulated upon IL-27 stimulation, and recombinant CXCL10 and CXCL11 inhibited FGF-2-induced proliferation in vitro. siRNA targeting of STAT1 almost completely abrogated CXCL10 and CXCL11 expression as well as the proliferative...

  10. Effect of metformin on proliferation and related genes expression of human osteoblast MG63 under high glucose



    Objective To study the effect of metformin on proliferation and related genes expression of human osteoblast.Methods The proliferation of MG63 cells under high glucose intervened with metformin was measured by CCK-8 assay. The activity of intracellular alkaline phosphatase

  11. Transcription factor genes essential for cell proliferation and replicative lifespan in budding yeast

    Kamei, Yuka; Tai, Akiko; Dakeyama, Shota; Yamamoto, Kaori; Inoue, Yamato; Kishimoto, Yoshifumi; Ohara, Hiroya; Mukai, Yukio, E-mail:


    Many of the lifespan-related genes have been identified in eukaryotes ranging from the yeast to human. However, there is limited information available on the longevity genes that are essential for cell proliferation. Here, we investigated whether the essential genes encoding DNA-binding transcription factors modulated the replicative lifespan of Saccharomyces cerevisiae. Heterozygous diploid knockout strains for FHL1, RAP1, REB1, and MCM1 genes showed significantly short lifespan. {sup 1}H-nuclear magnetic resonance analysis indicated a characteristic metabolic profile in the Δfhl1/FHL1 mutant. These results strongly suggest that FHL1 regulates the transcription of lifespan related metabolic genes. Thus, heterozygous knockout strains could be the potential materials for discovering further novel lifespan genes. - Highlights: • Involvement of yeast TF genes essential for cell growth in lifespan was evaluated. • The essential TF genes, FHL1, RAP1, REB1, and MCM1, regulate replicative lifespan. • Heterozygous deletion of FHL1 changes cellular metabolism related to lifespan.

  12. With current gene markers, presymptomatic diagnosis of heritable disease is still a family affair


    In the last four years, genes or genetic markers have been identified for a host of disorders including Huntington's disease, cystic fibrosis, Duchenne muscular dystrophy, polycystic kidney disease, bipolar depressive disorder, retinoblastoma, Alzheimer's disease, and schizophrenia. Such discoveries have made it possible to diagnose in utero some 30 genetic diseases during the first trimester of pregnancy. Yet, while these newly discovered gene markers may be revolutionizing prenatal and presymptomatic diagnosis, they are in many respects halfway technology. Such was the opinion of several speakers at a conference sponsored by the American Medical Association in Washington, DC. At the conference, entitled DNA Probes in the Practice of Medicine, geneticists emphasized that gene markers - stretches of DNA that are usually inherited in tandem with a disease gene - are usually not sufficient for presymptomatic diagnosis of genetic disease in an individual.

  13. Development and mapping of SSR markers linked to resistance-gene homologue clusters in common bean

    Luz; Nayibe; Garzon; Matthew; Wohlgemuth; Blair


    Common bean is an important but often a disease-susceptible legume crop of temperate,subtropical and tropical regions worldwide. The crop is affected by bacterial, fungal and viral pathogens. The strategy of resistance-gene homologue(RGH) cloning has proven to be an efficient tool for identifying markers and R(resistance) genes associated with resistances to diseases. Microsatellite or SSR markers can be identified by physical association with RGH clones on large-insert DNA clones such as bacterial artificial chromosomes(BACs). Our objectives in this work were to identify RGH-SSR in a BAC library from the Andean genotype G19833 and to test and map any polymorphic markers to identify associations with known positions of disease resistance genes. We developed a set of specific probes designed for clades of common bean RGH genes and then identified positive BAC clones and developed microsatellites from BACs having SSR loci in their end sequences. A total of 629 new RGH-SSRs were identified and named BMr(bean microsatellite RGH-associated markers). A subset of these markers was screened for detecting polymorphism in the genetic mapping population DOR364 × G19833. A genetic map was constructed with a total of 264 markers,among which were 80 RGH loci anchored to single-copy RFLP and SSR markers. Clusters of RGH-SSRs were observed on most of the linkage groups of common bean and in positions associated with R-genes and QTL. The use of these new markers to select for disease resistance is discussed.

  14. Demethylation of FANCF gene may be a potential treatment through inhibiting the proliferation of cervical cancer

    Min Li; Chanyu Zhang


    Objective: The aim of the study was to explore the effect of demethylating agent 5-Aza-2'-deoxycytidine (5-ADC) on expression of Fanconi anemia complementation group F (FANCF) gene and the proliferation of cervical cancer cells, to observe cell's sensitivity to chemotherapeutic drug taxol, and to explore the antitumor effect of 5-ADC as well as the new treatment of cervical cancer. Methods: Cervical cancer cell lines SiHa (FANCF gene full-methylated) and Hela (unmethylated) were treated with 5-ADC. We used the methylation-specific PCR (MSP), reverse transcription-polymerase chain reaction (RT-PCR) and Western blot to detect the FANCF methylation, mRNA and protein respectively. The 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the proliferation of cells. The cytotoxicity of taxol was measured by flow cytometer. The nude mice bearing SiHa was used to observe the effect of 5-ADC in vivo. Results: Inhibition of DNA promoter methylation by 5-ADC reactivated the expression of FANCF mRNA and protein in SiHa cells, consistent with decreased growth speed and increased taxol resistance. These results were proven in experiments in vivo. Conclusion: The 5-ADC probably become a potential treatment drug through inhibiting the proliferation of cervical cancer cells in taxol-resistant patients.

  15. Gene targeting in melanoma therapy: exploiting of surface markers and specific promoters

    Sverdlov E. D.


    Full Text Available One of the problems of gene therapy of melanoma is effective expression of therapeutic gene in tumor cells and their metastases but not in normal cells. In this review, we will consider a two-step approach to a highly specific gene therapy. At the first step, therapeutic genes are delivered specifically to tumor cells using cell surface markers of melanoma cells as targets. At the second step, a specific expression of the therapeutic genes in tumor cells is ensured. Surface markers of melanoma cells were analyzed as potential targets for therapeutic treatment. Criteria for choosing the most promising targets are proposed. The use of specific melanoma promoters allows to further increase the specificity of treatment via transcriptional control of therapeutic gene expression in melanoma cells.

  16. Mapping of Fertility Restoring Gene for Aegilops kotschyi Cytoplasmic Male Sterility in Wheat Using SSR Markers

    LIU Bao-shen; SUN Qi-xin; GAO Qing-rong; SUN Lan-zhen; XIE Chao-jie; LI Chuan-you; NI Zhong-fu; DOU Bing-de


    LK783 was found to be a good fertility restorer for K-type male sterility of wheat. Microsatellite markers were employed to map the major restoring gene in LK783. Maintainer and restorer DNA pools were established using the extreme sterile and fertile plants among (KJ5418A//911289/LK783)F1 population,respectively. Seventy-nine sets of SSR primers were screened for polymorphism between the two pools, 6 of which were found polymorphic. Linkage analysis showed that Xgwm11, Xgwm18 , Xgwm264a and Xgwm273were linked to the restoring gene in LK783, while Xgwm11, Xgwm18 and Xgwm273 were co-segregated. The distance between the Rf gene in LK783 and the three co-segregated markers was 6.54±4.37 cM, the distance between Rf gene and Xgwm264a was 5.71±4.10 cM. The four SSR markers were located on chromosome 1BS by amplifying the DNA of nulli-tetrasomics and ditelosomics of CS with the 4 sets of primers, indicating that the major restoring gene in LK783 was located on 1BS, but the relative location of the gene was different from Rfv1, allelism of the two genes should be further investigated. The breeding for new fertility restorer lines of K-type cytoplasmic male sterility in wheat would be facilitated by using the four polymorphic markers.

  17. FBXW7/hCDC4 controls glioma cell proliferation in vitro and is a prognostic marker for survival in glioblastoma patients

    Hagedorn Martin


    Full Text Available Abstract Background In the quest for novel molecular mediators of glioma progression, we studied the regulation of FBXW7 (hCDC4/hAGO/SEL10, its association with survival of patients with glioblastoma and its potential role as a tumor suppressor gene in glioma cells. The F-box protein Fbxw7 is a component of SCFFbxw7, a Skp1-Cul1-F-box E3 ubiquitin ligase complex that tags specific proteins for proteasome degradation. FBXW7 is mutated in several human cancers and functions as a haploinsufficient tumor suppressor in mice. Any of the identified targets, Cyclin E, c-Myc, c-Jun, Notch1/4 and Aurora-A may have oncogenic properties when accumulated in tumors with FBXW7 loss. Results We tested the expression of FBXW7 in human glioma biopsies by quantitative PCR and compared the transcript levels of grade IV glioma (glioblastoma, G-IV with those of grade II tumors (G-II. In more than 80% G-IV, expression of FBXW7 was significantly reduced. In addition, levels of FBXW7 were correlated with survival indicating a possible implication in tumor aggressiveness. Locus 4q31.3 which carries FBXW7 was investigated by in situ hybridization on biopsy touchprints. This excluded allelic loss as the principal cause for low expression of FBXW7 in G-IV tumors. Two targets of Fbxw7, Aurora-A and Notch4 were preferentially immunodetected in G-IV biopsies. Next, we investigated the effects of FBXW7 misregulation in glioma cells. U87 cells overexpressing nuclear isoforms of Fbxw7 lose the expression of the proliferation markers PCNA and Ki-67, and get counterselected in vitro. This observation fits well with the hypothesis that Fbxw7 functions as a tumor suppressor in astroglial cells. Finally, FBXW7 knockdown in U87 cells leads to defects in mitosis that may promote aneuploidy in progressing glioma. Conclusion Our results show that FBXW7 expression is a prognostic marker for patients with glioblastoma. We suggest that loss of FBXW7 plays an important role in glioma

  18. PDPN gene promotes the proliferation of immature Bovine Sertoli cells in vitro.

    Gao, Yi; Qin, Lihong; Yang, Yuwei; Dong, Xue; Zhao, Zijiao; Zhang, Guoliang; Zhao, Zhihui


    Podoplanin (PDPN) is a transmembrane receptor which is involved in various physiological and pathological processes, such as cell motility, invasion, tumor metastasis and blood vessels formation. Although there are reports on the involvement of PDPN in Sertoli cells in human and mice, the role of PDPN on the development of bovine Sertoli cells has not been reported. In the present study, Sertoli cells were isolated from 1-day-old bovine testes by two steps enzyme digestion method. Feulgen staining of satellite karyosomes and inhibin immunofluorescence staining suggested that the isolated immature Sertoli cells were very pure. Transfection with overexpression plasmid pBI-CMV3-PDPN and interference shRNA plasmid indicated that PDPN could significantly promote Sertoli cells cycle progression, cells proliferation and androgen-binding protein (ABP) production. Our results indicated that PDPN gene plays a significant role in the proliferation and maturation of bovine Sertoli cells.

  19. Identification of Molecular Marker Linked to Salt Tolerance Gene in Alfalfa


    The study has established the F2 offspring obtained by crossing salt-tolerant with salt-sensitive alfalfa, and appraised the salt-tolerant F2 offspring seedling was evaluated in pot culture. With the F2 segregated population, the research has obtained a molecular marker linked with salt-tolerant genes of alfalfa using the improved BSA combined with RAPD. The RAPD PCR products were excised from the agarose gel and purified using a kit, then were mixed with pMD-18T vector and sequenced. Sequencing result indicated the RAPD marker was 1 438 bp in length. Similarity researches using blast in Genbank indicated that the nucleotide sequence of the RAPD marker showed 93% and 91% similarity with mth2-6el8 gene fragment (347 bp) and mth2-33122 gene fragment (334 bp) of Medicago truncatula respectively. Medicago truncatula is a close relative of alfalfa and Mth2-6e18 is a molecular marker of the gene coding for a cysteine protease which was salt inducible in some plants. These results indicated the RAPD marker was possibly related to cysteine protease genes in alfalfa.

  20. Candidate Gene Identification with SNP Marker-Based Fine Mapping of Anthracnose Resistance Gene Co-4 in Common Bean.

    Burt, Andrew J; William, H Manilal; Perry, Gregory; Khanal, Raja; Pauls, K Peter; Kelly, James D; Navabi, Alireza


    Anthracnose, caused by Colletotrichum lindemuthianum, is an important fungal disease of common bean (Phaseolus vulgaris). Alleles at the Co-4 locus confer resistance to a number of races of C. lindemuthianum. A population of 94 F4:5 recombinant inbred lines of a cross between resistant black bean genotype B09197 and susceptible navy bean cultivar Nautica was used to identify markers associated with resistance in bean chromosome 8 (Pv08) where Co-4 is localized. Three SCAR markers with known linkage to Co-4 and a panel of single nucleotide markers were used for genotyping. A refined physical region on Pv08 with significant association with anthracnose resistance identified by markers was used in BLAST searches with the genomic sequence of common bean accession G19833. Thirty two unique annotated candidate genes were identified that spanned a physical region of 936.46 kb. A majority of the annotated genes identified had functional similarity to leucine rich repeats/receptor like kinase domains. Three annotated genes had similarity to 1, 3-β-glucanase domains. There were sequence similarities between some of the annotated genes found in the study and the genes associated with phosphoinositide-specific phosphilipases C associated with Co-x and the COK-4 loci found in previous studies. It is possible that the Co-4 locus is structured as a group of genes with functional domains dominated by protein tyrosine kinase along with leucine rich repeats/nucleotide binding site, phosphilipases C as well as β-glucanases.

  1. Tagging and Utilization Bruchid Resistance Gene Using PCR Markers in Mungbean

    CHENG Xu-zhen; WANG Su-hua; WU Shao-yu; ZHOU Ji-hong; WANG Shu-min; Charles Y Yang


    Sixteen mungbean lines were analyzed using 56 random primers. Different DNA bands were detected between Bruchid resistant lines and susceptible lines. According to the cluster results, the 16 lines can be divided into four groups,including brucid resistant wild types, resistant cultivated lines, resistant progenies and a mixed group. BSA method was used to identify DNA markers that related with bruchid resistant gene by using resistant line and susceptible line and their F2 progeny. One codominant marker was identified, which generated a fragment of 1.79 kb in resistant lines and 1.03kb in susceptible lines. Finally, this codominant marker was considered to be tightly linked with bruchid resistant gene and could be useful in resistant germplasm identification and marker-assisted selection.

  2. Incorporating gene co-expression network in identification of cancer prognosis markers

    Li Yang


    Full Text Available Abstract Background Extensive biomedical studies have shown that clinical and environmental risk factors may not have sufficient predictive power for cancer prognosis. The development of high-throughput profiling technologies makes it possible to survey the whole genome and search for genomic markers with predictive power. Many existing studies assume the interchangeability of gene effects and ignore the coordination among them. Results We adopt the weighted co-expression network to describe the interplay among genes. Although there are several different ways of defining gene networks, the weighted co-expression network may be preferred because of its computational simplicity, satisfactory empirical performance, and because it does not demand additional biological experiments. For cancer prognosis studies with gene expression measurements, we propose a new marker selection method that can properly incorporate the network connectivity of genes. We analyze six prognosis studies on breast cancer and lymphoma. We find that the proposed approach can identify genes that are significantly different from those using alternatives. We search published literature and find that genes identified using the proposed approach are biologically meaningful. In addition, they have better prediction performance and reproducibility than genes identified using alternatives. Conclusions The network contains important information on the functionality of genes. Incorporating the network structure can improve cancer marker identification.

  3. [Use of genes of carbon metabolism enzymes as molecular markers of Chlorobi Phylum representatives].

    Turova, T P; Kovaleva, O L; Gorlenko, V M; Ivanovskiĭ, R N


    This work examined the feasibility of using certain genes of carbon metabolism enzymes as molecular markers adequate for studying phylogeny and ecology of green sulfur bacteria (GSB) of the Chlorobi phylum. Primers designed to amplify the genes of ATP citrate lyase (aclB) and citrate synthase (gltA) revealed the respective genes in the genomes of all of the newly studied GSB strains. The phylogenetic trees constructed based on nucleotide sequences of these genes and amino acid sequences of the conceptually translated proteins were on the whole congruent with the 16S rRNA gene tree, with the single exception of GltA of Chloroherpeton thalassium, which formed a separate branch beyond the cluster comprised by other representatives of the Chlorobi phylum. Thus, the aclB genes but not gltA genes proved to be suitable for the design of primers specific to all Chlorobi representatives. Therefore, it was the aclB gene that was further used asa molecular marker to detect GSB in enrichment cultures and environmental samples. AclB phylotypes of GSB were revealed in all of the samples studied, with the exception of environmental samples from soda lakes. The identification of the revealed phylotypes was in agreement with the identification based on the FMO protein gene (fmo), is a well-known Chlorobi-specific molecular marker.

  4. Gene transcripts as potential diagnostic markers for allergic contact dermatitis

    Hansen, Malene Barré; Skov, Lone; Menné, Torkil;


    The standard procedure for diagnosing allergic contact dermatitis is to perform a patch test. Because this has several disadvantages, the development of a new in vitro test system would be of immense value. Gene transcripts that distinguish allergics from non-allergics may have the potential...

  5. Inhibitory effects of PIN1 antisense gene on the proliferation of human osteosarcoma cells


    Objective: To evaluate the inhibitory effects of PIN1 antisense gene on the proliferation of human osteosarcoma cells. Methods: Different doses of antisense PIN1 gene (0,20,50,100,200,250μl) were transfected into osteosarcoma MG-63 cells. The cells and the culture supernatants before and after transfection were collected. The cell growth curve was made using MTT method. The cell growth cycle and apoptosis were detected by FCM. The expression of PIN1 was detected by Western blot. The expression of PIN1 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Results: MTT and FCM assays indicated that the transfection of antisense PIN1 gene could inhibit proliferation of MG-63 cells and lead to cell apoptosis. Western-blot assays revealed the MG-63 cells transfected with antisense PIN1 gene had weaker expression than those without transfection with antisense PIN1 gene, and the band intensity was negatively related with doses. The cells transfected with different doses of gene (0,20,50,100,200,250 μl) had different absorbance rate(0.854 ± 0.136,0. 866 ± 0. 138,0. 732 ± 0. 154, 0. 611 ± 0. 121,0. 547 ± 0. 109,0. 398 ± 0. 113,0. 320 ± 0. 151 ), with significant difference assessed by F and q test ( P < 0.05). The absorbance rate of PINI mRNA was 0. 983 ± 0.125,0.988 ± 0.127, 0.915 ± 0.157,0.786 ± 0.125,0.608 ± 0.124,0.433 ± 0.130,0.410 ± 0. 158 respectively ( P < 0.05). Conclusion: The expression of PINlmRNA in MG-63 cells could be inhibited by antisense PIN1 gene, and then the expression of PIN1 was reduced and depressed, and so the proliferation of human osteosarcoma cells MG-63 was inhibited.

  6. Inhibition of Proliferation of Human Osteosarcoma Cells Transfected with PIN1 Antisense Gene

    XIONG Wenhua; CHEN Anmin; GUO Fengjin


    Objective: To evaluate the inhibition of proliferation of human osteosarcoma cells transfected with Pin1 anti-sense gene. Methods: Different doses of antisense Pin1 gene (0, 20, 50, 100, 200, 250μL) were transfected into osteosarcoma MG-63 cells. The cells and culture supernatant before and after transfection were collected. The curve of cell growth was made by MTT method. The cell growth cycle and apoptosis were detected by FCM. The expression of Pin1 was detected by Western-blot and that of Pin1 mRNA by polymerase chain reaction (RT-PCR) respectively. Results: MTT and FCM assays indicated that the transfection by antisense Pin1 gene could inhibit MG-63 proliferation and induce apoptosis. Western-blot assays revealed that the antisense Pin1 gene-transfected MG-63 cells had weaker staining than those without transfected with antisense Pin1 gene, and staining intensity was negatively related with doses. The cells transfected by different doses of gene (0, 20, 50, 100, 200, 250μL) had different absorbance rate: 0.854±0.136, 0.866±0.138, 0.732±0.154, 0.611±0.121, 0.547±0.109, 0.398±0.113,0.320±0.151 respectively, with the difference being significant by F and q test (P<0.05). The expression of Pin1 mRNA had the similar results and its absorbance rate was 0.983±0.125, 0.988±0.127, 0.915±0.157,0.786±0.125, 0.608±0.124, 0.433±0.130, 0.410±0.158 respectively (P<0.05). Conclusion: The expression of Pin1 mRNA in MG-63 cells could be inhibited by antisense Pin1 gene, so to reduce the expression of Pin1 and depress the proliferation of human osteosarcoma cells MG-63.

  7. Identification of molecular markers linked to rice bacterial blight resistance genes from Oryza meyeriana

    Jing WANG,Chen CHENG,Yanru ZHOU,Yong YANG,Qiong MEI,Junmin LI,Ye CHENG,Chengqi YAN,Jianping CHEN


    Full Text Available Y73 is a progeny of asymmetric somatic hybridization between Oryza sativa cv. Dalixiang and the wild rice species Oryza meyeriana. Inoculation with a range of strains of Xanthomonas oryzae pv. oryzae showed that Y73 had inherited a high level of resistance to rice bacterial blight (BB from its wild parent. An F2 population of 7125 individuals was constructed from the cross between Y73 and a BB-susceptible cultivar IR24. After testing 615 SSR and STS markers covering the 12 rice chromosomes, 186 markers were selected that showed polymorphism between Y73 and IR24. Molecular markers linked to the BB resistance genes in Y73 were scanned using the F2 population and the polymorphic markers. The SSR marker RM128 on chromosome 1, the STS marker R03D159 on chromosome 3 and the STS marker R05D104 on chromosome 5 were found to be linked to the rice BB resistance genes in Y73.

  8. PL1 fusion gene: a novel visual selectable marker gene that confers tolerance to multiple abiotic stresses in transgenic tomato.

    Jin, Feng; Li, Shu; Dang, Lijie; Chai, Wenting; Li, Pengli; Wang, Ning Ning


    Visual selectable markers, including the purple color caused by the accumulation of anthocyanins, have been proposed for use as antibiotic-free alternatives. However, the excessive accumulation of anthocyanins seriously inhibits the growth and development of transgenic plants. In our study, the AtDWF4 promoter from Arabidopsis and the tomato LeANT1 gene, encoding a MYB transcription factor, were used to construct the PL1 fusion gene to test whether it could be used as a visual selectable marker gene for tomato transformation. All the PL1 transgenic shoots exhibited intense purple color on shoot induction medium. In the transgenic tomato plants, PL1 was highly expressed in the cotyledons, but expressed only slightly in the true leaves and other organs. The expression of PL1 had no significantly adverse effects on the growth or development of the transgenic tomato plants, and conferred tolerance to multiple abiotic stresses in them. With the “cut off green shoots” method, multiple independent 35S::GFP transgenic tomato lines were successfully obtained using PL1 as the selectable marker gene. These results suggest that PL1 has potential application of visual selectable marker gene for tomato transformation.

  9. Peroxisome proliferator-activated receptor-gamma gene: a key regulator of adipocyte differentiation in chickens.

    Wang, Y; Mu, Y; Li, H; Ding, N; Wang, Q; Wang, Y; Wang, S; Wang, N


    The peroxisome proliferator-activated receptors (PPAR) are members of the nuclear hormone receptor superfamily. Peroxisome proliferator-activated receptor-gamma is regarded as a "master regulator" of adipocyte differentiation in mammals. The current study was designed to investigate the function and regulatory mechanism of PPARgamma in chicken adipogenesis by RNA interference. Preadipocytes were isolated from the abdominal fat tissue of 12-d-old chickens and cultured. Small-interference PPARgamma RNA (siPPARgamma) was synthesized by in vitro transcription and transfected into chicken preadipocytes by using liposomes. The suppressive effect of siPPARgamma was detected by real-time reverse-transcription PCR and reverse-transcription PCR. The results showed that transient transfection with siPPARgamma significantly inhibited differentiation and enhanced proliferation of chicken preadipocytes (P adipogenesis-associated adipocyte fatty acid-binding protein gene was down-regulated when PPARgamma was silenced. The current work indicates that PPARgamma is a key regulator of chicken preadipocyte differentiation.

  10. Advances in Localization and Molecular Markers of Wheat Leaf Rust Resistance Genes

    YANG Wen-xiang; LIU Da-qun


    Genetic resistance is the most economical method of reducing yield losses caused by wheat leaf rust. To identify the leaf rust resistance genes in commonly used parental germplasm and released cultivars become very important for utilizing the genetic resistance tc wheat leaf rust fully. Up to date, about 90 leaf rust resistance genes have been found,of which 51 genes have been located and mapped to special chromosomes, and 56 genes have been designated officially according to the standards set forth in the Catalogue of Gene Symbols for wheat. Twenty-four wheat leaf rust resistance genes have been developed for their molecular markers. It is very important to isolate, characterize, and map leaf rust resistance genes due to the resistance losses of the genes caused by the pathogen continuously.

  11. Identification of molecular markers linked to the mildew resistance gene Pl-d in apple.

    James, C M; Clarke, J B; Evans, K M


    Powdery mildew poses a serious problem for apple growers, and resistance to the disease is a major objective in breeding programmes for cultivar improvement. As selective pressure allows pathogens to overcome previously reliable resistances, there is a need for the introduction of novel resistance genes into new breeding lines. This investigation is concerned with the identification of the first set of molecular markers linked to the gene for mildew resistance, Pl-d, from the accession 'D12'. As no prior information on the map position or markers for Pl-d were available, a bulked-segregant approach was used to test 49 microsatellite primers, 176 amplified fragment length polymorphism (AFLP) primers and 80 random amplified polymorphic DNA (RAPD) primers in a progeny segregating for Pl-d resistance, 'Fiesta' (susceptible) x A871-14 ('Worcester Pearmain' x 'D12'). The segregations of the markers identified in the resistant and susceptible bulks were scored in the progeny, then the recombination fractions between Pl-d and the most tightly linked markers were calculated and a map prepared. Three AFLP, one RAPD and two microsatellite markers were identified. One AFLP was developed into a sequence-characterised amplified region marker, while the microsatellites CH03C02 and CH01D03 were flanking markers, 7 and 11 recombination units, respectively, from Pl-d. Two more distant microsatellites on the same linkage group, CH01D09 and CH01G12, confirmed the orientation of the markers on the linkage group. These microsatellites place Pl-d on the bottom of linkage group 12 in published apple maps, a region where a number of other disease resistance genes have been identified.

  12. Molecular mapping of the Pinus monticola Cr2 gene using AFLP and SCAR markers

    A.K.M. Ekramoddoullah


    Full Text Available White pine blister rust (WPBR, caused by Cronartium ribicola, is a devastating disease in five-needle pines. Genetic resistance is an important component of integrated strategies to control WPBR. The major resistance gene Cr2, discovered by Kinloch etal.(1999, is also effective against British Columbia (BC isolates of WPBR (Hunt et al. 2004. Pyramiding Cr2 gene with other resistancegenes is being pursued as a strategy in BC white pine breeding. To facilitate this strategy, we have recently identified a few RAPD markerslinked to Cr2 at one side (Liu et al. 2006. The objective of the present study was to identify amplified fragment length polymorphism(AFLP markers linked to both sides of Cr2 for its more precise apping. Use of the AFLP technique combined with bulked segregant analysis (BSA and haploid segregation analysis allowed the identification of five AFLP markers. Of these five AFLP markers in the Cr2 linkage, markers EacccMccgat-365, EactgMcccac- 290, and EacagEacag-750 werelinked in coupling and EacagMcccag-160r and EacccMccgat-180r in repulsion. Following cloning and sequencing of the AFLP andRAPD markers, specific PCR primers were designed and used in the amplification of sequence characterized amplified region(SCAR markers at both sides of Cr2. EacccMccgat- 365 and RAPD marker U570-843 reported previously were converted into SCARmarkers. These two SCARs segregated in a 1:1 (presence:absence ratio and the scoring cosegregated with their respective AFLP orRAPD marker. The SCAR marker EacccMccgat- 365-scar was positioned at 3.1 Kosambi cM from one side of Cr2 and U570-843-scarlocalized at 1.4 Kosambi cM from other side. Both SCAR markers can be useful in breeding programs with marker-assisted selection procedureto screen for resistance. This study represents the first report of the development of PCR-based sequence-specific markers linkedto blister rust resistance in five-needle pines. These findings may

  13. Molecular mapping of the Pinus monticola Cr2 gene using AFLP and SCAR markers

    A.K.M. Ekramoddoullah


    Full Text Available White pine blister rust (WPBR, caused by Cronartium ribicola, is a devastating disease in five-needle pines. Genetic resistance is an important component of integrated strategies to control WPBR. The major resistance gene Cr2, discovered by Kinloch etal.(1999, is also effective against British Columbia (BC isolates of WPBR (Hunt et al. 2004. Pyramiding Cr2 gene with other resistancegenes is being pursued as a strategy in BC white pine breeding. To facilitate this strategy, we have recently identified a few RAPD markerslinked to Cr2 at one side (Liu et al. 2006. The objective of the present study was to identify amplified fragment length polymorphism(AFLP markers linked to both sides of Cr2 for its more precise apping. Use of the AFLP technique combined with bulked segregant analysis (BSA and haploid segregation analysis allowed the identification of five AFLP markers. Of these five AFLP markers in the Cr2 linkage, markers EacccMccgat-365, EactgMcccac- 290, and EacagEacag-750 werelinked in coupling and EacagMcccag-160r and EacccMccgat-180r in repulsion. Following cloning and sequencing of the AFLP andRAPD markers, specific PCR primers were designed and used in the amplification of sequence characterized amplified region(SCAR markers at both sides of Cr2. EacccMccgat- 365 and RAPD marker U570-843 reported previously were converted into SCARmarkers. These two SCARs segregated in a 1:1 (presence:absence ratio and the scoring cosegregated with their respective AFLP orRAPD marker. The SCAR marker EacccMccgat- 365-scar was positioned at 3.1 Kosambi cM from one side of Cr2 and U570-843-scarlocalized at 1.4 Kosambi cM from other side. Both SCAR markers can be useful in breeding programs with marker-assisted selection procedureto screen for resistance. This study represents the first report of the development of PCR-based sequence-specific markers linkedto blister rust resistance in five-needle pines. These findings may

  14. Rheumatoid arthritis and pigmented villonodular synovitis: comparative analysis of cell polyploidy, cell cycle phases and expression of macrophage and fibroblast markers in proliferating synovial cells.

    Berger, I; Weckauf, H; Helmchen, B; Ehemann, V; Penzel, R; Fink, B; Bernd, L; Autschbach, F


    Rheumatoid arthritis (RA) and pigmented villonodular synovitis (PVNS) are aggressive diseases with progressive joint destruction. The present study aims to define cell cycle phases, polyploidy and the immunophenotype of proliferating synovial cells in both diseases. Synovial tissues from patients with proliferative-active RA, localized and diffuse PVNS were analysed by DNA flow cytometry, immunohistochemistry and double immunofluorescence with confocal laser scan microscopy. Expression of macrophage markers (CD68/CD163), fibroblast markers (h4Ph/CD55) and Ki67 antigen was examined. Synovial cells positive for either macrophage or fibroblast markers as well as double-labelled cells were found in both RA and PVNS. In RA, CD68/CD163+ synoviocytes were preferentially located in the vicinity of the synovial lining layer, while they were more randomly distributed in PVNS. Of cases with diffuse PVNS, 20% showed an aneuploid cell pattern. All samples of localized PVNS and RA were diploid. Proliferative activity was significantly higher in aneuploid PVNS. In spite of their histologically homogeneous appearance, proliferating synovial cells display a heterogeneous immunophenotype in both RA and PVNS, indicating functional properties of both macrophages and fibroblasts. Aneuploidy seems to be a special feature of diffuse PVNS.

  15. Nuclear factor XIIIa staining (clone AC-1A1 mouse monoclonal) is a sensitive and specific marker to discriminate sebaceous proliferations from other cutaneous clear cell neoplasms.

    Uhlenhake, Elizabeth E; Clark, Lindsey N; Smoller, Bruce R; Shalin, Sara C; Gardner, Jerad M


    Sebaceous carcinoma is a rare but serious malignancy that may be difficult to diagnose when poorly differentiated. Other epithelial tumors with clear cell change may mimic sebaceous carcinoma. Few useful or specific immunohistochemical markers for sebaceous differentiation are available. Nuclear staining with factor XIIIa (clone AC-1A1) was recently found to be a highly sensitive marker of sebaceous differentiation. We evaluated nuclear factor XIIIa (AC-1A1) staining in sebaceous neoplasms vs. other cutaneous clear cell tumors. We stained 27 sebaceous proliferations: sebaceous hyperplasia (7), sebaceous adenoma (8), sebaceoma (5), sebaceous carcinoma (7). We also stained 67 tumors with clear cell change: basal cell carcinoma (8), squamous cell carcinoma (8), hidradenoma (7), desmoplastic trichilemmoma (2), trichilemmoma (10), trichilemmal carcinoma (3), clear cell acanthoma (9), atypical fibroxanthoma (1), syringoma (8), trichoepithelioma (1), metastatic renal cell carcinoma (2), and nevi with balloon cell change (8). Nuclear factor XIIIa (AC-1A1) staining was present in 100% of sebaceous proliferations; 96% displayed strong staining. Non-sebaceous clear cell tumors were negative or only weakly positive with factor XIIIa (AC-1A1) in 95.5%; only 4.5% showed strong staining. This suggests that strong nuclear factor XIIIa (AC-1A1) staining is a sensitive and specific marker of sebaceous neoplasms vs. other clear cell tumors.

  16. Interaction between Calpain 5, Peroxisome proliferator-activated receptor-gamma and Peroxisome proliferator-activated receptor-delta genes: a polygenic approach to obesity

    Ruiz Agustín


    Full Text Available Abstract Context Obesity is a multifactorial disorder, that is, a disease determined by the combined effect of genes and environment. In this context, polygenic approaches are needed. Objective To investigate the possibility of the existence of a crosstalk between the CALPAIN 10 homologue CALPAIN 5 and nuclear receptors of the peroxisome proliferator-activated receptors family. Design Cross-sectional, genetic association study and gene-gene interaction analysis. Subjects The study sample comprise 1953 individuals, 725 obese (defined as body mass index ≥ 30 and 1228 non obese subjects. Results In the monogenic analysis, only the peroxisome proliferator-activated receptor delta (PPARD gene was associated with obesity (OR = 1.43 [1.04–1.97], p = 0.027. In addition, we have found a significant interaction between CAPN5 and PPARD genes (p = 0.038 that reduces the risk for obesity in a 55%. Conclusion Our results suggest that CAPN5 and PPARD gene products may also interact in vivo.

  17. Developmentally regulated site-specific marker gene excision in transgenic B. napus plants.

    Kopertekh, Lilya; Broer, Inge; Schiemann, Joachim


    We have developed a self-excision Cre-vector to remove marker genes from Brassica napus. In this vector cre recombinase gene and bar expression cassette were inserted between two lox sites in direct orientation. These lox-flanked sequences were placed between the seed-specific napin promoter and the gene of interest (vstI). Tissue-specific cre activation resulted in simultaneous excision of the recombinase and marker genes. The vector was introduced into B. napus by Agrobacterium-mediated transformation. F1 progeny of seven lines with single and multiple transgene insertions was subjected to segregation and molecular analysis. Marker-free plants could be detected and confirmed by PCR and Southern blot in all transgenic lines tested. The recombination efficiency expressed as a ratio of plants with complete gene excision to the total number of investigated plants varied from 13 to 81% dependent on the transgene copy number. Potential application of this system would be the establishment of marker-free transgenic plants in generatively propagated species.

  18. Epidemiology and gene markers of ulcerative colitis in the Chinese

    Jun Yun; Chang-Tai Xu; Bo-Rong Pan


    Inflammatory bowel disease (IBD) includes two similar yet distinct conditions called ulcerative colitis (UC) and Crohn's disease (CD). These diseases affect the digestive system and cause the inflammation of intestinal tissue, form sores and bleed easily. Most children with IBD arediagnosed in late childhood and adolescence. However, both UC and CD have been reported as early as in infancy. Most information pertaining to the epidemiology of IBD is based upon adult studies. Symptoms include abdominal pain, cramping, fatigue and diarrhea. Genetic factors play a significant role in determining IBD susceptibility. Epidemiological data support a genetic contribution to the pathogenesis of IBD. Recently, numerous new genes have been identified as being involved in the genetic susceptibility to IBD: TNF- 308A, CARD15 ( NOD2), MIF-173, N-acetyltransferase 2 ( NAT2), NKG2D (natural killer cell 2D), STAT6 (signal transducer and activator of transcription 6), CTLA-4 (cytotoxic T lymphocyte antigen-4), MICA-MICB (major histocompatibility complex A and B), HLA-DRB1, HLA class-?, IL-18, IL-4, MICA-A5, CD14, TLR4, Fas-670, p53 and NF-kB. The characterization of these novel genes has the potential to identify therapeutic agents and aid clinical assessment of phenotype and prognosis in patients with IBD (UC and CD).

  19. Cytosolic phospholipase A{sub 2} gene in human and rat: Chromosomal localization and polymorphic markers

    Tay, A.; Simon, J.S.; Jacob, H.J. [Univ. of Toronto (Canada)] [and others


    The authors report the chromosomal localization and a simple sequence repeat (SSR) in the cytosolic phospholipase A{sub 2} (cPLA{sub 2}) gene in both human and rat. A (CA){sub 18} repeat in the promoter of the rat gene was determined to exhibit length polymorphism when analyzed using the polymerase chain reaction (PCR) in 19 different inbred rat strains. Genotyping for this marker in 234 F{sub 2} progeny of a SHRXBN intercross mapped the gene to rat chromosome 13. Using a PCR strategy, a fragment of the promoter for the human gene was isolated, and a (CA){sub 18} repeat was identified. Since this marker displayed a low heterozygosity index, they also identified a mononucleotide repeat in the promoter for cPLA{sub 2} that displayed a polymorphism information content value of 0.76. The human gene was mapped using fluorescence in situ hybridization (FISH) to chromosome 1q25. Of interest, the gene encoding the enzyme prostaglandin-endoperoxide synthase 2 (cyclooxygenase-2), which acts on the arachidonic acid product of cPLA{sub 2}, was previously localized to this same chromosomal region, raising the possibility of coordinate regulation. Identification of intragenic markers may facilitate studies of polymorphic variants of these genes as candidates for disorders in which perturbations of the eicosanoid cascade may play a role. 20 refs., 3 figs., 2 tabs.

  20. CP2 gene as a useful viability marker for Cryptosporidium parvum.

    Lee, Soo-Ung; Joung, Migyo; Ahn, Myoung-Hee; Huh, Sun; Song, Hyunje; Park, Woo-Yoon; Yu, Jae-Ran


    The validity of the CP2 gene of Cryptosporidium parvum as a viability marker was evaluated using absolute quantitative real-time polymerase chain reaction (qPCR) assays. Total ribonucleic acid (RNA) was isolated from live and heat-killed C. parvum oocysts, and complementary deoxyribonucleic acid was synthesized and used as a template. The most accurate number of viable C. parvum oocysts was predicted when the CP2 gene was used as a target gene. The lower detection limit of the CP2 gene was ten oocysts, which was the most sensitive among examined target genes. With heat shock induction, only hsp70 messenger RNA (mRNA) was induced, and the predicted viable oocyst number was increased by heat shock for this marker. The CP2, hsp70, Cryptosporidium oocyst wall protein, and beta-tubulin mRNAs were not detected in heat-killed oocysts, but the 18S ribosomal ribonucleic acid (rRNA) showed heat stability until 48 h after heat killing. Although the 18S rRNA demonstrated the fastest response in crossing point (CP) value among the examined primer sets in qPCR, overestimation of viable oocysts was noted in the analysis with this gene. In conclusion, the CP2 gene was identified as the most sensitive, reliable, and accurate candidate of a viability marker of C. parvum by qPCR evaluation.

  1. Functional dissection of HOXD cluster genes in regulation of neuroblastoma cell proliferation and differentiation.

    Yunhong Zha

    Full Text Available Retinoic acid (RA can induce growth arrest and neuronal differentiation of neuroblastoma cells and has been used in clinic for treatment of neuroblastoma. It has been reported that RA induces the expression of several HOXD genes in human neuroblastoma cell lines, but their roles in RA action are largely unknown. The HOXD cluster contains nine genes (HOXD1, HOXD3, HOXD4, and HOXD8-13 that are positioned sequentially from 3' to 5', with HOXD1 at the 3' end and HOXD13 the 5' end. Here we show that all HOXD genes are induced by RA in the human neuroblastoma BE(2-C cells, with the genes located at the 3' end being activated generally earlier than those positioned more 5' within the cluster. Individual induction of HOXD8, HOXD9, HOXD10 or HOXD12 is sufficient to induce both growth arrest and neuronal differentiation, which is associated with downregulation of cell cycle-promoting genes and upregulation of neuronal differentiation genes. However, induction of other HOXD genes either has no effect (HOXD1 or has partial effects (HOXD3, HOXD4, HOXD11 and HOXD13 on BE(2-C cell proliferation or differentiation. We further show that knockdown of HOXD8 expression, but not that of HOXD9 expression, significantly inhibits the differentiation-inducing activity of RA. HOXD8 directly activates the transcription of HOXC9, a key effector of RA action in neuroblastoma cells. These findings highlight the distinct functions of HOXD genes in RA induction of neuroblastoma cell differentiation.

  2. Characterization and molecular marker screening of a rice bacteria-resistant gene Xa-min(t)

    CHEN Yan; HU Jun; QIAN Wei; TIAN Yingchuan; HE Chaozu


    To test the resistant spectrum of the Xa-min(t) gene introgressed from Oryza minuta, thirty-four isolates of different bacterial blight pathogen, Xanthomonas oryzae pv. oryzae (Xoo), from 11 countries were used to inoculate the Xa-min(t) introgression line 78-15. Four rice cultivars, IR24, C64 (IRBB21), Nipponbare and Zhonghua 11 were used as controls. The results showed that the Xa-min(t) gene was broad-spectrum and highly resistant to diverse Xoo isolates. The methods of bulk segregant analysis (BSA), randomly amplified polymorphic DNA (RAPD) and sequence characterized amplified regions (SCAR) were used to analyze F2 individuals of the hybrid IR24×78-15 and molecular genetic markers linked to Xa-min(t) gene were identified. A total of 800 arbitrary decamer oligonucleotide primers were used for RAPD analysis. Two RAPD markers, BE05300 and BE061400, produced by primers BE05 and BE06 respectively, were closely linked to the Xa-min(t) gene. Based on the sequences of these two markers, sequence specific primers were designed and used to screen all F2 plants. One RAPD marker, BE05300, was converted into a stable SCAR marker (ScBE05300). Linkage analysis was carried out using markers ScBE05300 and BE061400 on 948 and 719 F2 individuals of the hybrid IR24×78-15. Our results indicate that the genetic distances from Xa-min(t) to ScBE05300 and BE061400 are 2.2 cM and 3.7 cM respectively on the same side. This study may facilitate the construction of the fine physical map of the Xa-min(t) gene.

  3. Novel Implant Coating Agent Promotes Gene Expression of Osteogenic Markers in Rats during Early Osseointegration

    Kostas Bougas


    Full Text Available The aim of this study was to evaluate the early bone response around laminin-1-coated titanium implants. Forty-five rats distributed in three equally sized groups were provided with one control (turned and one test (laminin-1-coated implant and were sacrificed after 3, 7, and 21 days. Real-time reverse-transcriptase polymerase chain reaction was performed for osteoblast markers (alkaline phosphatase, runt-related transcription factor 2, osteocalcin, type I collagen, and bone morphogenic protein 2, osteoclast markers (cathepsin K and tartrate-resistant acid phosphatase, inflammation markers (tumor necrosis factor α, interleukin 1β and interleukin 10, and integrin β1. Bone implant contact (BIC and bone area (BA were assessed and compared to the gene expression. After 3 days, the expression of bone markers was higher for the control group. After 7 days, the expression of integrin β1 and osteogenic markers was enhanced for the test group, while cathepsin K and inflammation markers were down-regulated. No significant differences in BIC or BA were detected between test and control at any time point. As a conclusion, implant coating with laminin-1 altered gene expression in the bone-implant interface. However, traditional evaluation methods, as histomorphometry, were not adequately sensitive to detect such changes due to the short follow-up time.

  4. Haplotypes of the porcine peroxisome proliferator-activated receptor delta gene are associated with backfat thickness

    Blöcker Helmut


    Full Text Available Abstract Background Peroxisome proliferator-activated receptor delta belongs to the nuclear receptor superfamily of ligand-inducible transcription factors. It is a key regulator of lipid metabolism. The peroxisome proliferator-activated receptor delta gene (PPARD has been assigned to a region on porcine chromosome 7, which harbours a quantitative trait locus for backfat. Thus, PPARD is considered a functional and positional candidate gene for backfat thickness. The purpose of this study was to test this candidate gene hypothesis in a cross of breeds that were highly divergent in lipid deposition characteristics. Results Screening for genetic variation in porcine PPARD revealed only silent mutations. Nevertheless, significant associations between PPARD haplotypes and backfat thickness were observed in the F2 generation of the Mangalitsa × Piétrain cross as well as a commercial German Landrace population. Haplotype 5 is associated with increased backfat in F2 Mangalitsa × Piétrain pigs, whereas haplotype 4 is associated with lower backfat thickness in the German Landrace population. Haplotype 4 and 5 carry the same alleles at all but one SNP. Interestingly, the opposite effects of PPARD haplotypes 4 and 5 on backfat thickness are reflected by opposite effects of these two haplotypes on PPAR-δ mRNA levels. Haplotype 4 significantly increases PPAR-δ mRNA levels, whereas haplotype 5 decreases mRNA levels of PPAR-δ. Conclusion This study provides evidence for an association between PPARD and backfat thickness. The association is substantiated by mRNA quantification. Further studies are required to clarify, whether the observed associations are caused by PPARD or are the result of linkage disequilibrium with a causal variant in a neighbouring gene.

  5. Proliferating cell nuclear antigen (Pcna) as a direct downstream target gene of Hoxc8

    Min, Hyehyun; Lee, Ji-Yeon; Bok, Jinwoong; Chung, Hyun Joo [Department of Anatomy, Embryology Laboratory, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Kim, Myoung Hee, E-mail: [Department of Anatomy, Embryology Laboratory, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of)


    Hoxc8 is a member of Hox family transcription factors that play crucial roles in spatiotemporal body patterning during embryogenesis. Hox proteins contain a conserved 61 amino acid homeodomain, which is responsible for recognition and binding of the proteins onto Hox-specific DNA binding motifs and regulates expression of their target genes. Previously, using proteome analysis, we identified Proliferating cell nuclear antigen (Pcna) as one of the putative target genes of Hoxc8. Here, we asked whether Hoxc8 regulates Pcna expression by directly binding to the regulatory sequence of Pcna. In mouse embryos at embryonic day 11.5, the expression pattern of Pcna was similar to that of Hoxc8 along the anteroposterior body axis. Moreover, Pcna transcript levels as well as cell proliferation rate were increased by overexpression of Hoxc8 in C3H10T1/2 mouse embryonic fibroblast cells. Characterization of 2.3 kb genomic sequence upstream of Pcna coding region revealed that the upstream sequence contains several Hox core binding sequences and one Hox-Pbx binding sequence. Direct binding of Hoxc8 proteins to the Pcna regulatory sequence was verified by chromatin immunoprecipitation assay. Taken together, our data suggest that Pcna is a direct downstream target of Hoxc8.

  6. Stanniocalcin-2 is a HIF-1 target gene that promotes cell proliferation in hypoxia

    Law, Alice Y.S. [Department of Biology, Hong Kong Baptist University, Kowloon Tong (Hong Kong); Wong, Chris K.C., E-mail: [Department of Biology, Hong Kong Baptist University, Kowloon Tong (Hong Kong)


    Stanniocalcin-2 (STC2), the paralog of STC1, has been suggested as a novel target of oxidative stress response to protect cells from apoptosis. The expression of STC2 has been reported to be highly correlated with human cancer development. In this study, we reported that STC2 is a HIF-1 target gene and is involved in the regulation of cell proliferation. STC2 was shown to be up-regulated in different breast and ovarian cancer cells, following exposure to hypoxia. Using ovarian cancer cells (SKOV3), the underlying mechanism of HIF-1 mediated STC2 gene transactivation was characterized. Hypoxia-induced STC2 expression was found to be HIF-1{alpha} dependent and required the recruitment of p300 and HDAC7. Using STC2 promoter deletion constructs and site-directed mutagenesis, two authentic consensus HIF-1 binding sites were identified. Under hypoxic condition, the silencing of STC2 reduced while the overexpression of STC2 increased the levels of phosphorylated retinoblastoma and cyclin D in both SKOV3 and MCF7 cells. The change in cell cycle proteins correlated with the data of the serial cell counts. The results indicated that cell proliferation was reduced in STC2-silenced cells but was increased in STC2-overexpressing hypoxic cells. Solid tumor progression is usually associated with hypoxia. The identification and functional analysis of STC2 up-regulation by hypoxia, a feature of the tumor microenvironment, sheds light on a possible role for STC2 in tumors.

  7. DNACLUST: accurate and efficient clustering of phylogenetic marker genes

    Liu Bo


    Full Text Available Abstract Background Clustering is a fundamental operation in the analysis of biological sequence data. New DNA sequencing technologies have dramatically increased the rate at which we can generate data, resulting in datasets that cannot be efficiently analyzed by traditional clustering methods. This is particularly true in the context of taxonomic profiling of microbial communities through direct sequencing of phylogenetic markers (e.g. 16S rRNA - the domain that motivated the work described in this paper. Many analysis approaches rely on an initial clustering step aimed at identifying sequences that belong to the same operational taxonomic unit (OTU. When defining OTUs (which have no universally accepted definition, scientists must balance a trade-off between computational efficiency and biological accuracy, as accurately estimating an environment's phylogenetic composition requires computationally-intensive analyses. We propose that efficient and mathematically well defined clustering methods can benefit existing taxonomic profiling approaches in two ways: (i the resulting clusters can be substituted for OTUs in certain applications; and (ii the clustering effectively reduces the size of the data-sets that need to be analyzed by complex phylogenetic pipelines (e.g., only one sequence per cluster needs to be provided to downstream analyses. Results To address the challenges outlined above, we developed DNACLUST, a fast clustering tool specifically designed for clustering highly-similar DNA sequences. Given a set of sequences and a sequence similarity threshold, DNACLUST creates clusters whose radius is guaranteed not to exceed the specified threshold. Underlying DNACLUST is a greedy clustering strategy that owes its performance to novel sequence alignment and k-mer based filtering algorithms. DNACLUST can also produce multiple sequence alignments for every cluster, allowing users to manually inspect clustering results, and enabling more

  8. ETS-related transcription factors ETV4 and ETV5 are involved in proliferation and induction of differentiation-associated genes in embryonic stem (ES) cells.

    Akagi, Tadayuki; Kuure, Satu; Uranishi, Kousuke; Koide, Hiroshi; Costantini, Frank; Yokota, Takashi


    The pluripotency and self-renewal capacity of embryonic stem (ES) cells is regulated by several transcription factors. Here, we show that the ETS-related transcription factors Etv4 and Etv5 (Etv4/5) are specifically expressed in undifferentiated ES cells, and suppression of Oct3/4 results in down-regulation of Etv4/5. Simultaneous deletion of Etv4 and Etv5 (Etv4/5 double knock-out (dKO)) in ES cells resulted in a flat, epithelial cell-like appearance, whereas the morphology changed into compact colonies in a 2i medium (containing two inhibitors for GSK3 and MEK/ERK). Expression levels of self-renewal marker genes, including Oct3/4 and Nanog, were similar between wild-type and dKO ES cells, whereas proliferation of Etv4/5 dKO ES cells was decreased with overexpression of cyclin-dependent kinase inhibitors (p16/p19, p15, and p57). A differentiation assay revealed that the embryoid bodies derived from Etv4/5 dKO ES cells were smaller than the control, and expression of ectoderm marker genes, including Fgf5, Sox1, and Pax3, was not induced in dKO-derived embryoid bodies. Microarray analysis demonstrated that stem cell-related genes, including Tcf15, Gbx2, Lrh1, Zic3, and Baf60c, were significantly repressed in Etv4/5 dKO ES cells. The artificial expression of Etv4 and/or Etv5 in Etv4/5 dKO ES cells induced re-expression of Tcf15 and Gbx2. These results indicate that Etv4 and Etv5, potentially through regulation of Gbx2 and Tcf15, are involved in the ES cell proliferation and induction of differentiation-associated genes in ES cells.

  9. Gene expression profiling of craniofacial fibrous dysplasia reveals ADAMTS2 overexpression as a potential marker

    Zhou, Shang-Hui; Yang, Wen-Jun; Liu, Sheng-Wen; Li, Jiang; Zhang, Chun-Ye; Zhu, Yun; Zhang, Chen-Ping


    Fibrous dysplasia (FD) as an abnormal bone growth is one of the common fibro-osseous leasions (FOL) in oral and maxillofacial region, however, its etiology still remains unclear. Here, we performed gene expression profiling of FD using microarray analysis to explore the key molecule events in FD development, and develop potential diagnostic markers or therapeutic targets for FD. We found that 1,881 genes exhibited differential expression with more than two-fold changes in FD compared to norma...

  10. Detecting RAPD Markers Linked to Ripe Rot Resistance Genes in Chinese Wild Vitis

    WANG Yue-jin; XU Yan; ZHANG Jian-xia; ZHOU Peng; WAN Yi-zhen


    With F1 individuals of the cross combination 88-110 of 83-4-96 ( V. quinquangularis Rehd. )× Muscat Rose ( V. vinifera L. ), the RAPD marker OPC15-1300 linked to ripe rot ( Gloeosporium fruetigenum Berk. ) resistance genes in Chinese wild Vitis was gained using bulked segregation analysis(BSA). And it was found that OPC15-1300 could be hereditary from the resistant parent (83-4-96) after the marker was tested in 50 F1 plants of the cross combination 88-110, 32 accessions of 8 Chinese wild Vitis species and 14cultivars of V. vinifera L. Also, it has provided a solid basis for molecular marker-assisted selection (MAS)and for possibly cloning disease resistance genes in the future.

  11. Physiological evaluation of the filamentous fungus Trichoderma reesei in production processes by marker gene expression analysis

    Penttilä Merja


    Full Text Available Abstract Background Biologically relevant molecular markers can be used in evaluation of the physiological state of an organism in biotechnical processes. We monitored at high frequency the expression of 34 marker genes in batch, fed-batch and continuous cultures of the filamentous fungus Trichoderma reesei by the transcriptional analysis method TRAC (TRanscript analysis with the aid of Affinity Capture. Expression of specific genes was normalised either with respect to biomass or to overall polyA RNA concentration. Expressional variation of the genes involved in various process relevant cellular functions, such as protein production, growth and stress responses, was related to process parameters such as specific growth and production rates and substrate and dissolved oxygen concentrations. Results Gene expression of secreted cellulases and recombinant Melanocarpus albomyces laccase predicted the trends in the corresponding extracellular enzyme production rates and was highest in a narrow "physiological window" in the specific growth rate (μ range of 0.03 – 0.05 h-1. Expression of ribosomal protein mRNAs was consistent with the changes in μ. Nine starvation-related genes were found as potential markers for detection of insufficient substrate feed for maintaining optimal protein production. For two genes induced in anaerobic conditions, increasing transcript levels were measured as dissolved oxygen decreased. Conclusion The data obtained by TRAC supported the usefulness of focused and intensive transcriptional analysis in monitoring of biotechnical processes providing thus tools for process optimisation purposes.

  12. Gastrointestinal differentiation marker Cytokeratin 20 is regulated by homeobox gene CDX1

    Chan, Carol W M; Wong, Newton A; Liu, Ying


    -expression analysis and other approaches. Keratin 20 (KRT20), a member of the intermediate filament and a well-known marker of intestinal differentiation, was initially identified as one of the genes likely to be directly regulated by CDX1. CDX1 and KRT20 mRNA expression were significantly correlated in a panel of 38...

  13. Systems Biology in Animal Breeding: Identifying relationships among markers, genes, and phenotypes

    The Breeding and Genetics Symposium titled “Systems Biology in Animal Breeding: Identifying relationships among markers, genes, and phenotypes” was held at the Joint Annual Meeting of the American Dairy Science Association and the American Society of Animal Science in Phoenix, AZ, July 15 to 19, 201...

  14. LAPTM4B Gene Expression And Polymorphism As Diagnostic Markers Of Breast Cancer In Egyptian Patients

    Shaker Olfat


    Full Text Available Background: The aim of this study was to investigate the association between LAPTM4B gene polymorphism and the risk of breast cancer among Egyptian female patients. Also, measurement was done of its serum level to evaluate its significance as a diagnostic marker for breast cancer.

  15. Transcriptional and posttranscriptional regulation of the proliferating cell nuclear antigen gene

    Chang, C.D.; Ottavio, L.; Travali, S.; Lipson, K.E.; Baserga, R. (Temple Univ., Philadelphia, PA (USA). School of Medicine)


    The steady-state mRNA levels of the proliferating cell nuclear antigen (PCNA) gene are growth regulated. In a previous paper the authors reported that introns (especially intron 4) participate in growth regulation of the PCNA gene. They have now investigated the role of the 5{prime}-flanking sequence of the human PCNA gene stably transfected into BALB/c 3T3 cells. Promoters of different lengths (from{minus}2856 to {minus}45 upstream of the cap site) were tested. All promoters except the Aatll promoter ({minus}45), including a short HpaII promoter ({minus}210), were sufficient for a response to serum, platelet-derived growth factor, and to a lesser extent epidermal growth factor. No construct responded to insulin or platelet-poor plasma. The AatII promoter had little detectable activity. Transcriptional activity was also determined in BALB/c 3T3 cells carrying various constructs of the human PCNA gene by two methods: run-on transcription and reverse transcription-polymerase chain reaction (the latter measuring the heterogeneous nuclear RNA (hnRNA) steady-state levels).

  16. Microcephaly gene links Trithorax and REST/NRSF to control neural stem cell proliferation and differentiation

    Yang, Yawei J.; Baltus, Andrew E.; Mathew, Rebecca S.; Murphy, Elisabeth A.; Evrony, Gilad D.; Gonzalez, Dilenny M.; Wang, Estee P.; Marshall-Walker, Christine A.; Barry, Brenda J.; Murn, Jernej; Tatarakis, Antonis; Mahajan, Muktar A.; Samuels, Herbert H.; Shi, Yang; Golden, Jeffrey A.; Mahajnah, Muhammad; Shenhav, Ruthie; Walsh, Christopher A.


    SUMMARY Microcephaly is a neurodevelopmental disorder causing significantly reduced cerebral cortex size. Many known microcephaly gene products localize to centrosomes, regulating cell fate and proliferation. Here, we identify and characterize a nuclear zinc finger protein, ZNF335/NIF-1, as a causative gene for severe microcephaly, small somatic size, and neonatal death. Znf335-null mice are embryonically lethal and conditional knockout leads to severely reduced cortical size. RNA-interference and postmortem human studies show that Znf335 is essential for neural progenitor self-renewal, neurogenesis, and neuronal differentiation. ZNF335 is a component of a vertebrate-specific, trithorax H3K4-methylation complex, directly regulating REST/NRSF, a master regulator of neural gene expression and cell fate, as well as other essential neural-specific genes. Our results reveal ZNF335 as an essential link between H3K4 complexes and REST/NRSF, and provide the first direct genetic evidence that this pathway regulates human neurogenesis and neuronal differentiation. PMID:23178126

  17. The Novel Gene CRNDE Encodes a Nuclear Peptide (CRNDEP Which Is Overexpressed in Highly Proliferating Tissues.

    Lukasz Michal Szafron

    Full Text Available CRNDE, recently described as the lncRNA-coding gene, is overexpressed at RNA level in human malignancies. Its role in gametogenesis, cellular differentiation and pluripotency has been suggested as well. Herein, we aimed to verify our hypothesis that the CRNDE gene may encode a protein product, CRNDEP. By using bioinformatics methods, we identified the 84-amino acid ORF encoded by one of two CRNDE transcripts, previously described by our research team. This ORF was cloned into two expression vectors, subsequently utilized in localization studies in HeLa cells. We also developed a polyclonal antibody against CRNDEP. Its specificity was confirmed in immunohistochemical, cellular localization, Western blot and immunoprecipitation experiments, as well as by showing a statistically significant decrease of endogenous CRNDEP expression in the cells with transient shRNA-mediated knockdown of CRNDE. Endogenous CRNDEP localizes predominantly to the nucleus and its expression seems to be elevated in highly proliferating tissues, like the parabasal layer of the squamous epithelium, intestinal crypts or spermatocytes. After its artificial overexpression in HeLa cells, in a fusion with either the EGFP or DsRed Monomer fluorescent tag, CRNDEP seems to stimulate the formation of stress granules and localize to them. Although the exact role of CRNDEP is unknown, our preliminary results suggest that it may be involved in the regulation of the cell proliferation. Possibly, CRNDEP also participates in oxygen metabolism, considering our in silico results, and the correlation between its enforced overexpression and the formation of stress granules. This is the first report showing the existence of a peptide encoded by the CRNDE gene.

  18. Application of GFAT as a novel selection marker to mediate gene expression.

    Guogan Wu

    Full Text Available The enzyme glutamine: fructose-6-phosphate aminotransferase (GFAT, also known as glucosamine synthase (GlmS, catalyzes the formation of glucosamine-6-phosphate from fructose-6-phosphate and is the first and rate-limiting enzyme of the hexosamine biosynthetic pathway. For the first time, the GFAT gene was proven to possess a function as an effective selection marker for genetically modified (GM microorganisms. This was shown by construction and analysis of two GFAT deficient strains, E. coli ΔglmS and S. pombe Δgfa1, and the ability of the GFAT encoding gene to mediate plasmid selection. The gfa1 gene of the fission yeast Schizosaccharomyces pombe was deleted by KanMX6-mediated gene disruption and the Cre-loxP marker removal system, and the glmS gene of Escherichia coli was deleted by using λ-Red mediated recombinase system. Both E. coli ΔglmS and S. pombe Δgfa1 could not grow normally in the media without addition of glucosamine. However, the deficiency was complemented by transforming the plasmids that expressed GFAT genes. The xylanase encoding gene, xynA2 from Thermomyces lanuginosus was successfully expressed and secreted by using GFAT as selection marker in S. pombe. Optimal glucosamine concentration for E. coli ΔglmS and S. pombe Δgfa1 growth was determined respectively. These findings provide an effective technique for the construction of GM bacteria without an antibiotic resistant marker, and the construction of GM yeasts to be applied to complex media.

  19. Candidate Gene Identification with SNP Marker-Based Fine Mapping of Anthracnose Resistance Gene Co-4 in Common Bean

    Burt, Andrew J.; William, H. Manilal; Perry, Gregory; Khanal, Raja; Pauls, K. Peter; Kelly, James D.; Navabi, Alireza


    Anthracnose, caused by Colletotrichum lindemuthianum, is an important fungal disease of common bean (Phaseolus vulgaris). Alleles at the Co–4 locus confer resistance to a number of races of C. lindemuthianum. A population of 94 F4:5 recombinant inbred lines of a cross between resistant black bean genotype B09197 and susceptible navy bean cultivar Nautica was used to identify markers associated with resistance in bean chromosome 8 (Pv08) where Co–4 is localized. Three SCAR markers with known linkage to Co–4 and a panel of single nucleotide markers were used for genotyping. A refined physical region on Pv08 with significant association with anthracnose resistance identified by markers was used in BLAST searches with the genomic sequence of common bean accession G19833. Thirty two unique annotated candidate genes were identified that spanned a physical region of 936.46 kb. A majority of the annotated genes identified had functional similarity to leucine rich repeats/receptor like kinase domains. Three annotated genes had similarity to 1, 3-β-glucanase domains. There were sequence similarities between some of the annotated genes found in the study and the genes associated with phosphoinositide-specific phosphilipases C associated with Co-x and the COK–4 loci found in previous studies. It is possible that the Co–4 locus is structured as a group of genes with functional domains dominated by protein tyrosine kinase along with leucine rich repeats/nucleotide binding site, phosphilipases C as well as β-glucanases. PMID:26431031

  20. Candidate Gene Identification with SNP Marker-Based Fine Mapping of Anthracnose Resistance Gene Co-4 in Common Bean.

    Andrew J Burt

    Full Text Available Anthracnose, caused by Colletotrichum lindemuthianum, is an important fungal disease of common bean (Phaseolus vulgaris. Alleles at the Co-4 locus confer resistance to a number of races of C. lindemuthianum. A population of 94 F4:5 recombinant inbred lines of a cross between resistant black bean genotype B09197 and susceptible navy bean cultivar Nautica was used to identify markers associated with resistance in bean chromosome 8 (Pv08 where Co-4 is localized. Three SCAR markers with known linkage to Co-4 and a panel of single nucleotide markers were used for genotyping. A refined physical region on Pv08 with significant association with anthracnose resistance identified by markers was used in BLAST searches with the genomic sequence of common bean accession G19833. Thirty two unique annotated candidate genes were identified that spanned a physical region of 936.46 kb. A majority of the annotated genes identified had functional similarity to leucine rich repeats/receptor like kinase domains. Three annotated genes had similarity to 1, 3-β-glucanase domains. There were sequence similarities between some of the annotated genes found in the study and the genes associated with phosphoinositide-specific phosphilipases C associated with Co-x and the COK-4 loci found in previous studies. It is possible that the Co-4 locus is structured as a group of genes with functional domains dominated by protein tyrosine kinase along with leucine rich repeats/nucleotide binding site, phosphilipases C as well as β-glucanases.

  1. Fine Mapping for Identification of Citrus Alternaria Brown Spot Candidate Resistance Genes and Development of New SNP Markers for Marker-Assisted Selection

    Cuenca, Jose; Aleza, Pablo; Garcia-Lor, Andres; Ollitrault, Patrick; Navarro, Luis


    Alternaria brown spot (ABS) is a serious disease affecting susceptible citrus genotypes, which is a strong concern regarding citrus breeding programs. Resistance is conferred by a recessive locus (ABSr) previously located by our group within a 3.3 Mb genome region near the centromere in chromosome III. This work addresses fine-linkage mapping of this region for identifying candidate resistance genes and develops new molecular markers for ABS-resistance effective marker-assisted selection (MAS). Markers closely linked to ABSr locus were used for fine mapping using a 268-segregating diploid progeny derived from a heterozygous susceptible × resistant cross. Fine mapping limited the genomic region containing the ABSr resistance gene to 366 kb, flanked by markers at 0.4 and 0.7 cM. This region contains nine genes related to pathogen resistance. Among them, eight are resistance (R) gene homologs, with two of them harboring a serine/threonine protein kinase domain. These two genes along with a gene encoding a S-adenosyl-L-methionine-dependent-methyltransferase protein, should be considered as strong candidates for ABS-resistance. Moreover, the closest SNP was genotyped in 40 citrus varieties, revealing very high association with the resistant/susceptible phenotype. This new marker is currently used in our citrus breeding program for ABS-resistant parent and cultivar selection, at diploid, triploid and tetraploid level. PMID:28066498

  2. W::Neo: a novel dual-selection marker for high efficiency gene targeting in Drosophila.

    Wenke Zhou

    Full Text Available We have recently developed a so-called genomic engineering approach that allows for directed, efficient and versatile modifications of Drosophila genome by combining the homologous recombination (HR-based gene targeting with site-specific DNA integration. In genomic engineering and several similar approaches, a "founder" knock-out line must be generated first through HR-based gene targeting, which can still be a potentially time and resource intensive process. To significantly improve the efficiency and success rate of HR-based gene targeting in Drosophila, we have generated a new dual-selection marker termed W::Neo, which is a direct fusion between proteins of eye color marker White (W and neomycin resistance (Neo. In HR-based gene targeting experiments, mutants carrying W::Neo as the selection marker can be enriched as much as fifty times by taking advantage of the antibiotic selection in Drosophila larvae. We have successfully carried out three independent gene targeting experiments using the W::Neo to generate genomic engineering founder knock-out lines in Drosophila.

  3. Biological pathways, candidate genes and molecular markers associated with quality-of-life domains: an update

    Sprangers, Mirjam A.G.; Thong, Melissa S.Y.; Bartels, Meike; Barsevick, Andrea; Ordoñana, Juan; Shi, Qiuling; Wang, Xin Shelley; Klepstad, Pål; Wierenga, Eddy A.; Singh, Jasvinder A.; Sloan, Jeff A.


    Background There is compelling evidence of a genetic foundation of patient-reported QOL. Given the rapid development of substantial scientific advances in this area of research, the current paper updates and extends reviews published in 2010. Objectives The objective is to provide an updated overview of the biological pathways, candidate genes and molecular markers involved in fatigue, pain, negative (depressed mood) and positive (well-being/happiness) emotional functioning, social functioning, and overall QOL. Methods We followed a purposeful search algorithm of existing literature to capture empirical papers investigating the relationship between biological pathways and molecular markers and the identified QOL domains. Results Multiple major pathways are involved in each QOL domain. The inflammatory pathway has the strongest evidence as a controlling mechanism underlying fatigue. Inflammation and neurotransmission are key processes involved in pain perception and the COMT gene is associated with multiple sorts of pain. The neurotransmitter and neuroplasticity theories have the strongest evidence for their relationship with depression. Oxytocin-related genes and genes involved in the serotonergic and dopaminergic pathways play a role in social functioning. Inflammatory pathways, via cytokines, also play an important role in overall QOL. Conclusions Whereas the current findings need future experiments and replication efforts, they will provide researchers supportive background information when embarking on studies relating candidate genes and/or molecular markers to QOL domains. The ultimate goal of this area of research is to enhance patients’ QOL. PMID:24604075

  4. Haploid Origin of Cork Oak Anther Embryos Detected by Enzyme and RAPD Gene Markers.

    Bueno; Agundez; Gomez; Carrascosa; Manzanera


    In vitro-induced cork oak (Quercus suber L.) embryos from anther cultures proved to be of haploid origin both by enzyme and RAPD gene marker analysis. The problem considered was to ascertain if embryo cultures originated either from a single haploid cell, from a microspore, or from multiple haploid cells. Therefore, a heterozygotic gene was searched for in the parent tree. The gene coding for shikimate dehydrogenase (SKDH1) proved to be heterozygous in the parental tree, and subsequently, these allozymes were screened for the embryos induced in anther cultures from the same tree. Only haploid embryos were found, confirming the microspore origin. Different genotypes were not identified inside each anther by isozyme analysis, probably because of selective pressure for one embryo early in development, but both parental SKDH1 alleles were found in the embryos of different anthers. The banding patterns detected by RAPD markers permitted the identification of multiple microspore origins inside each anther.

  5. Development of universal genetic markers based on single-copy orthologous (COSII) genes in Poaceae.

    Liu, Hailan; Guo, Xiaoqin; Wu, Jiasheng; Chen, Guo-Bo; Ying, Yeqing


    KEY MESSAGE : We develop a set of universal genetic markers based on single-copy orthologous (COSII) genes in Poaceae. Being evolutionary conserved, single-copy orthologous (COSII) genes are particularly useful in comparative mapping and phylogenetic investigation among species. In this study, we identified 2,684 COSII genes based on five sequenced Poaceae genomes including rice, maize, sorghum, foxtail millet, and brachypodium, and then developed 1,072 COSII markers whose transferability and polymorphism among five bamboo species were further evaluated with 46 pairs of randomly selected primers. 91.3 % of the 46 primers obtained clear amplification in at least one bamboo species, and 65.2 % of them produced polymorphism in more than one species. We also used 42 of them to construct the phylogeny for the five bamboo species, and it might reflect more precise evolutionary relationship than the one based on the vegetative morphology. The results indicated a promising prospect of applying these markers to the investigation of genetic diversity and the classification of Poaceae. To ease and facilitate access of the information of common interest to readers, a web-based database of the COSII markers is provided ( ).

  6. Noncoding RNA small nucleolar RNA host gene 1 promote cell proliferation in nonsmall cell lung cancer

    J You


    Full Text Available Background: Nonsmall cell lung cancer (NSCLC is the major cause of cancer death worldwide. Increasing evidence shows that noncoding RNAs (ncRNAs are widely involved in the development and progression of NSCLC. ncRNA small nucleolar RNA host gene 1 (SNHG1 has not been studied in cancer, especially its role in lung cancer remains unknown. Our studies were designed to investigate the expression and biological significance of SNHG1 in lung cancer. SNHG1 may be a novel ncRNA in early diagnosis in lung cancer. Methods: Noncoding RNA SNHG1 expression in 7 lung cancer cell lines was measured by quantitative real-time polymerase chain reaction. RNA interference approaches were used to find the biological functions of SNHG1. The effect of SNHG1 on proliferation was evaluated by cell count and crystal violet stains. Results: Noncoding RNA SNHG1 expression was significantly upregulated in lung cancer cells when compared with normal bronchial epithelial cells. In addition, in vitro assays our results indicated that knockdown of SNHG1 inhibited cell proliferation. Conclusions: Our data indicated that ncRNA SNHG1 is significantly upregulated in NSCLC cell lines and may represent a new biomarker and a potential therapeutic target for NSCLC intervention.

  7. TDIF peptide signaling regulates vascular stem cell proliferation via the WOX4 homeobox gene in Arabidopsis.

    Hirakawa, Yuki; Kondo, Yuki; Fukuda, Hiroo


    The indeterminate nature of plant growth and development depends on the stem cell system found in meristems. The Arabidopsis thaliana vascular meristem includes procambium and cambium. In these tissues, cell-cell signaling, mediated by a ligand-receptor pair made of the TDIF (for tracheary element differentiation inhibitory factor) peptide and the TDR/PXY (for TDIF RECEPTOR/ PHLOEM INTERCALATED WITH XYLEM) membrane protein kinase, promotes proliferation of procambial cells and suppresses their xylem differentiation. Here, we report that a WUSCHEL-related HOMEOBOX gene, WOX4, is a key target of the TDIF signaling pathway. WOX4 is expressed preferentially in the procambium and cambium, and its expression level was upregulated upon application of TDIF in a TDR-dependent manner. Genetic analyses showed that WOX4 is required for promoting the proliferation of procambial/cambial stem cells but not for repressing their commitment to xylem differentiation in response to the TDIF signal. Thus, at least two intracellular signaling pathways that diverge after TDIF recognition by TDR might regulate independently the behavior of vascular stem cells. Detailed observations in loss-of-function mutants revealed that TDIF-TDR-WOX4 signaling plays a crucial role in the maintenance of the vascular meristem organization during secondary growth.

  8. Molecular marker assisted gene stacking for biotic and abiotic stress resistance genes in an elite rice cultivar.

    Das, Gitishree; Rao, G J N


    Severe yield loss due to various biotic stresses like bacterial blight (BB), gall midge (insect) and Blast (disease) and abiotic stresses like submergence and salinity are a serious constraint to the rice productivity throughout the world. The most effective and reliable method of management of the stresses is the enhancement of host resistance, through an economical and environmentally friendly approach. Through the application of marker assisted selection (MAS) technique, the present study reports a successful pyramidization of genes/QTLs to confer resistance/tolerance to blast (Pi2, Pi9), gall Midge (Gm1, Gm4), submergence (Sub1), and salinity (Saltol) in a released rice variety CRMAS2621-7-1 as Improved Lalat which had already incorporated with three BB resistance genes xa5, xa13, and Xa21 to supplement the Xa4 gene present in Improved Lalat. The molecular analysis revealed clear polymorphism between the donor and recipient parents for all the markers that are tagged to the target traits. The conventional backcross breeding approach was followed till BC3F1 generation and starting from BC1F1 onwards, marker assisted selection was employed at each step to monitor the transfer of the target alleles with molecular markers. The different BC3F1s having the target genes/QTLs were inter crossed to generate hybrids with all 10 stress resistance/tolerance genes/QTLs into a single plant/line. Homozygous plants for resistance/tolerance genes in different combinations were recovered. The BC3F3 lines were characterized for their agronomic and quality traits and promising progeny lines were selected. The SSR based background selection was done. Most of the gene pyramid lines showed a high degree of similarity to the recurrent parent for both morphological, grain quality traits and in SSR based background selection. Out of all the gene pyramids tested, two lines had all the 10 resistance/tolerance genes and showed adequate levels of resistance/tolerance against the five target

  9. Molecular Marker Assisted Selection for Yield-Enhancing Genes in the Progeny of Minghui63 × O.rufipogon

    WANG Yue-guang; WANG Bin; DENG Qi-yun; LIANG Feng-shan; XING Quan-hua; LI Ji-ming; XONG Yue-dong; SUN Shi-mong; GUO Bao-tai; YUAN Long-ping


    Two yield-enhancing genes (yldl.1 and yld2.1) are located on chromosomes 1 and 2 respectively in a weedy relative of cultivated rice, Oryza rufipogon. SSR markers RM9 and RM166 are closely linked with the two loci respectively. Minghui63 (MH63) has been a widely used restoration line in hybrid rice production in China during the past two decades. The F1 of cross "MH63 × O.rufipogon" was backcrossed with MH63 generation by generation. RM9 and RM166 were used to select the plants from the progeny of the backcross populations. The results were as follows: (1) In BC2F1 population, the percentage of the individuals which have RM9 and RM166 amplified bands simultaneously was 12.2%, while in the BC3F1 population, that was 16.3%. (2) Among 400individuals of BC3F1, four yield-promising plants were obtained, with yield being 30% more than that of MH63. (3) The products amplified by primer RM166 in O. rufipogon and MH63 were sequenced. It was found that the DNA fragment sequence amplified by RM166 from MH63 was 101 bp shorter than that from O. rufipogon. The 101 bp sequence is a part of an intron of the PCNA (proliferating cell nuclear antigen) gene.

  10. Differentially expressed genes in human peripheral blood as potential markers for statin response.

    Won, Hong-Hee; Kim, Suk Ran; Bang, Oh Young; Lee, Sang-Chol; Huh, Wooseong; Ko, Jae-Wook; Kim, Hyung-Gun; McLeod, Howard L; O'Connell, Thomas M; Kim, Jong-Won; Lee, Soo-Youn


    There is a considerable inter-individual variation in response to statin therapy and one third of patients do not meet their treatment goals. We aimed to identify differentially expressed genes that might be involved in the effects of statin treatment and to suggest potential markers to guide statin therapy. Forty-six healthy Korean subjects received atorvastatin; their whole-genome expression profiles in peripheral blood were analyzed before and after atorvastatin administration in relation with changes in lipid profiles. The expression patterns of the differentially expressed genes were also compared with the data of familial hypercholesterolemia (FH) patients and controls. Pairwise comparison analyses revealed differentially expressed genes involved in diverse biological processes and molecular functions related with immune responses. Atorvastain mainly affected antigen binding, immune or inflammatory response including interleukin pathways. Similar expression patterns of the genes were observed in patients with FH and controls. The Charcol-Leyden crystal (CLC), CCR2, CX3CR1, LRRN3, FOS, LDLR, HLA-DRB1, ERMN, and TCN1 genes were significantly associated with cholesterol levels or statin response. Interestingly, the CLC gene, which was significantly altered by atorvastatin administration and differentially expressed between FH patients and controls, showed much bigger change in high-responsive group than in low-responsive group. We identified differentially expressed genes that might be involved in mechanisms underlying the known pleiotropic effects of atorvastatin, baseline cholesterol levels, and drug response. Our findings suggest CLC as a new candidate marker for statin response, and further validation is needed.

  11. Depletion of p42.3 gene inhibits proliferation and invasion in melanoma cells.

    Liu, Hui; Zhu, Min; Li, Zhongwu; Wang, Yan; Xing, Rui; Lu, Youyong; Xue, Weicheng


    The p42.3 gene is identified recently, and the upregulated expression has been characterized in a variety of human cancers and embryonic tissues but not yet in malignant melanoma. In this study, we explored the role of p42.3 gene in the development of melanoma. The expression of p42.3 was detected by immunohistochemistry staining of 261 cases of patient lesions, including nevi and melanoma, and its correlation with clinical pathological characteristics and prognosis was analyzed. Furthermore, a series of in vitro assays were used to investigate the biological function of p42.3 in melanoma cells. Immunohistochemistry staining showed an elevated expression level of p42.3 in melanoma compared to nevi (P = 0.001). Statistical analysis indicated that this high level was well correlated with patients' clinical stage (P = 0.045), but not with gender, age, clinical type, mitotic rate, and overall survival (P > 0.05). Moreover, in vitro assays showed knockdown p42.3 gene expression could inhibit the biological profiling, including proliferation, migration, and invasion of melanoma cells, and also affect PI3K/Akt pathway, MAPK pathway, and β-catenin. This study suggests that p42.3, acting like an oncogene, is involved in the malignant transformation process of melanoma and may serve as a biomarker for diagnostic and treatment purposes.

  12. Germ cell nuclear factor directly represses the transcription of peroxisome proliferator-activated receptor delta gene

    Chengqiang He; Naizheng Ding; Jie Kang


    Germ cell nuclear factor (GCNF) is a transcription factor that can repress gene transcription and plays an important role during spermatogenesis. Peroxisome proliferator-activated receptor delta (PPARδ) is a nuclear hormone receptor belonging to the steroid receptor superfamily.It can activate the expression of many genes,including those involved in lipid metabolism.In this report,we showed that GCNF specifically interacts with PPARδ promoter.Overexpression of GCNF in African green monkey SV40 transformed kidney fibroblast COS7 cells and mouse embryo fibroblast NIH 3T3 cells represses the activity of PPARδ promoter.The mutation of GCNF response element in PPARδ promoter relieves the repression in NIH 3T3 cells and mouse testis.Moreover,we showed that GCNF in nuclear extracts of mouse testis is able to bind to PPARδ promoter directly.We also found that GCNF and PPARδ mRNA were expressed with different patterns in mouse testis by in situ hybridization.These results suggested that GCNF might be a negative regulator of PPARδ gene expression through its direct interaction with PPARδ promoter in mouse testis.

  13. Dietary tocopherols inhibit cell proliferation, regulate expression of ERα, PPARγ, and Nrf2, and decrease serum inflammatory markers during the development of mammary hyperplasia.

    Smolarek, Amanda K; So, Jae Young; Thomas, Paul E; Lee, Hong Jin; Paul, Shiby; Dombrowski, Anne; Wang, Chung-Xiou; Saw, Constance Lay-Lay; Khor, Tin Oo; Kong, Ah-Ng Tony; Reuhl, Kenneth; Lee, Mao-Jung; Yang, Chung S; Suh, Nanjoo


    Previous clinical and epidemiological studies of vitamin E have used primarily α-tocopherol for the prevention of cancer. However, γ-tocopherol has demonstrated greater anti-inflammatory and anti-tumor activity than α-tocopherol in several animal models of cancer. This study assessed the potential chemopreventive activities of a tocopherol mixture containing 58% γ-tocopherol (γ-TmT) in an established rodent model of mammary carcinogenesis. Female ACI rats were utilized due to their sensitivity to 17β-estradiol (E2 ) to induce mammary hyperplasia and neoplasia. The rats were implanted subcutaneously with sustained release E2 pellets and given dietary 0.3% or 0.5% γ-TmT for 2 or 10 wk. Serum E2 levels were significantly reduced by the treatment with 0.5% γ-TmT. Serum levels of inflammatory markers, prostaglandin E2 and 8-isoprostane, were suppressed by γ-TmT treatment. Histology of mammary glands showed evidence of epithelial hyperplasia in E2 -treated rats. Immunohistochemical analysis of the mammary glands revealed a decrease in proliferating cell nuclear antigen (PCNA), cyclooxygenase-2 (COX-2), and estrogen receptor α (ERα), while there was an increase in cleaved-caspase 3, peroxisome proliferator-activated receptor γ (PPARγ), and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in γ-TmT-treated rats. In addition, treatment with γ-TmT resulted in a decrease in the expression of ERα mRNA, whereas mRNA levels of ERβ and PPARγ were increased. In conclusion, γ-TmT was shown to suppress inflammatory markers, inhibit E2 -induced cell proliferation, and upregulate PPARγ and Nrf2 expression in mammary hyperplasia, suggesting that γ-TmT may be a promising agent for human breast cancer prevention.

  14. Constitutive expression of tdTomato protein as a cytotoxicity and proliferation marker for space radiation biology.

    Chishti, Arif A; Hellweg, Christine E; Berger, Thomas; Baumstark-Khan, Christa; Feles, Sebastian; Kätzel, Thorben; Reitz, Günther


    The radiation risk assessment for long-term space missions requires knowledge on the biological effectiveness of different space radiation components, e.g. heavy ions, on the interaction of radiation and other space environmental factors such as microgravity, and on the physical and biological dose distribution in the human body. Space experiments and ground-based experiments at heavy ion accelerators require fast and reliable test systems with an easy readout for different endpoints. In order to determine the effect of different radiation qualities on cellular proliferation and the biological depth dose distribution after heavy ion exposure, a stable human cell line expressing a novel fluorescent protein was established and characterized. tdTomato, a red fluorescent protein of the new generation with fast maturation and high fluorescence intensity, was selected as reporter of cell proliferation. Human embryonic kidney (HEK/293) cells were stably transfected with a plasmid encoding tdTomato under the control of the constitutively active cytomegalovirus (CMV) promoter (ptdTomato-N1). The stably transfected cell line was named HEK-ptdTomato-N1 8. This cytotoxicity biosensor was tested by ionizing radiation (X-rays and accelerated heavy ions) exposure. As biological endpoints, the proliferation kinetics and the cell density reached 100 h after irradiation reflected by constitutive expression of the tdTomato were investigated. Both were reduced dose-dependently after radiation exposure. Finally, the cell line was used for biological weighting of heavy ions of different linear energy transfer (LET) as space-relevant radiation quality. The relative biological effectiveness of accelerated heavy ions in reducing cellular proliferation peaked at an LET of 91 keV/μm. The results of this study demonstrate that the HEK-ptdTomato-N1 reporter cell line can be used as a fast and reliable biosensor system for detection of cytotoxic damage caused by ionizing radiation.

  15. Expression of selected pathway-marker genes in human urothelial cells exposed chronically to a non-cytotoxic concentration of monomethylarsonous acid

    Matthew Medeiros


    Full Text Available Bladder cancer has been associated with chronic arsenic exposure. Monomethylarsonous acid [MMA(III] is a metabolite of inorganic arsenic and has been shown to transform an immortalized urothelial cell line (UROtsa at concentrations 20-fold less than arsenite. MMA(III was used as a model arsenical to examine the mechanisms of arsenical-induced transformation of urothelium. A previous microarray analysis revealed only minor changes in gene expression at 1 and 2 months of chronic exposure to MMA(III, contrasting with substantial changes observed at 3 months of exposure. To address the lack of information between 2 and 3 months of exposure (the critical period of transformation, the expression of select pathway marker genes was measured by PCR array analysis on a weekly basis. Cell proliferation rate, anchorage-independent growth, and tumorigenicity in SCID mice were also assessed to determine the early, persistent phenotypic changes and their association with the changes in expression of these selected marker genes. A very similar pattern of alterations in these genes was observed when compared to the microarray results, and suggested that early perturbations in cell signaling cascades, immunological pathways, cytokine expression, and MAPK pathway are particularly important in driving malignant transformation. These results showed a strong association between the acquired phenotypic changes that occurred as early as 1–2 months of chronic MMA(III exposure, and the observed gene expression pattern that is indicative of the earliest stages in carcinogenesis.

  16. Rapid and targeted introgression of genes into popular wheat cultivars using marker-assisted background selection.

    Harpinder S Randhawa

    Full Text Available A marker-assisted background selection (MABS-based gene introgression approach in wheat (Triticum aestivum L. was optimized, where 97% or more of a recurrent parent genome (RPG can be recovered in just two backcross (BC generations. A four-step MABS method was developed based on 'Plabsim' computer simulations and wheat genome structure information. During empirical optimization of this method, double recombinants around the target gene were selected in a step-wise fashion during the two BC cycles followed by selection for recurrent parent genotype on non-carrier chromosomes. The average spacing between carrier chromosome markers was <4 cM. For non-carrier chromosome markers that flanked each of the 48 wheat gene-rich regions, this distance was approximately 12 cM. Employed to introgress seedling stripe rust (Puccinia striiformis f. sp. tritici resistance gene Yr15 into the spring wheat cultivar 'Zak', marker analysis of 2,187 backcross-derived progeny resulted in the recovery of a BC(2F(2ratio3 plant with 97% of the recurrent parent genome. In contrast, only 82% of the recurrent parent genome was recovered in phenotypically selected BC(4F(7 plants developed without MABS. Field evaluation results from 17 locations indicated that the MABS-derived line was either equal or superior to the recurrent parent for the tested agronomic characteristics. Based on these results, MABS is recommended as a strategy for rapidly introgressing a targeted gene into a wheat genotype in just two backcross generations while recovering 97% or more of the recurrent parent genotype.

  17. Development of Agrobacterium-mediated virus-induced gene silencing and performance evaluation of four marker genes in Gossypium barbadense.

    Jinhuan Pang

    Full Text Available Gossypiumbarbadense is a cultivated cotton species and possesses many desirable traits, including high fiber quality and resistance to pathogens, especially Verticilliumdahliae (a devastating pathogen of Gossypium hirsutum, the main cultivated species. These elite traits are difficult to be introduced into G. hirsutum through classical breeding methods. In addition, genetic transformation of G. barbadense has not been successfully performed. It is therefore important to develop methods for evaluating the function and molecular mechanism of genes in G. barbadense. In this study, we had successfully introduced a virus-induced gene silencing (VIGS system into three cultivars of G. barbadense by inserting marker genes into the tobacco rattle virus (TRV vector. After we optimized the VIGS conditions, including light intensity, photoperiod, seedling age and Agrobacterium strain, 100% of plants agroinfiltrated with the GaPDS silencing vector showed white colored leaves. Three other marker genes, GaCLA1, GaANS and GaANR, were employed to further test this VIGS system in G. barbadense. The transcript levels of the endogenous genes in the silenced plants were reduced by more than 99% compared to control plants; these plants presented phenotypic symptoms 2 weeks after inoculation. We introduced a fusing sequence fragment of GaPDS and GaANR gene silencing vectors into a single plant, which resulted in both photobleaching and brownish coloration. The extent of silencing in plants agroinfiltrated with fusing two-gene-silencing vector was consistent with plants harboring a single gene silencing vector. The development of this VIGS system should promote analysis of gene function in G. barbadense, and help to contribute desirable traits for breeding of G. barbadense and G. hirsutum.

  18. A SSR Marker for Leaf Rust Resistance Gene Lr19 in Wheat

    LI Xing; YANG Wen-xiang; LI Ya-ning; LIU Da-qun; YAN Hong-fei; MENG Qing-fang; ZHANG Ting


    Microsatellite was carried out in Thatcher, six near-isogenic lines and F2 progeny of TcLr19×Thatcher to develop molecular markers for leaf rust resistance gene Lr19. Thirteen primer pairs were screened, of which one primer pair Xgwm44 displayed polymorphsim in the population of TcLr19, Thatcher, and their F2 generations. One marker closed linked to Lr19 resistance trait was obtained, and was named Xgwm44139 bp with the genetic distance 0.9 cM. The research shows that Lr19 has more potential in marker-assisted breeding programs in wheat and provides a step stone for mapping genetic map, physical map and the eventual cloning.

  19. 3'UTR Shortening Potentiates MicroRNA-Based Repression of Pro-differentiation Genes in Proliferating Human Cells.

    Yonit Hoffman


    Full Text Available Most mammalian genes often feature alternative polyadenylation (APA sites and hence diverse 3'UTR lengths. Proliferating cells were reported to favor APA sites that result in shorter 3'UTRs. One consequence of such shortening is escape of mRNAs from targeting by microRNAs (miRNAs whose binding sites are eliminated. Such a mechanism might provide proliferation-related genes with an expression gain during normal or cancerous proliferation. Notably, miRNA sites tend to be more active when located near both ends of the 3'UTR compared to those located more centrally. Accordingly, miRNA sites located near the center of the full 3'UTR might become more active upon 3'UTR shortening. To address this conjecture we performed 3' sequencing to determine the 3' ends of all human UTRs in several cell lines. Remarkably, we found that conserved miRNA binding sites are preferentially enriched immediately upstream to APA sites, and this enrichment is more prominent in pro-differentiation/anti-proliferative genes. Binding sites of the miR17-92 cluster, upregulated in rapidly proliferating cells, are particularly enriched just upstream to APA sites, presumably conferring stronger inhibitory activity upon shortening. Thus 3'UTR shortening appears not only to enable escape from inhibition of growth promoting genes but also to potentiate repression of anti-proliferative genes.


    韩为东; 赵亚力; 李琦; 母义明; 李雪; 宋海静; 陆祖谦


    Objective: Our previous studies have firstly demonstrated that 17(-E2 up-regulates LRP16 gene expression in human breast cancer MCF-7 cells, and ectopic expression of the LRP16 gene promotes MCF-7 cells proliferation. Here, the effects of the LRP16 gene expression on growth of MCF-7 human breast cancer cells and the mechanism were further studied by establishing two stably LRP16-inhibitory MCR-7 cell lines. Methods: Hairpin small interference RNA (siRNA) strategy, by which hairpin siRNA was released by U6 promoter and was mediated by pLPC-based retroviral vector, was adopted to knockdown endogenous LRP16 level in MCF-7 cells. And the hairpin siRNA against green fluorescence protein (GFP) was used as the negative control. The suppressant efficiency of the LRP16 gene expression was confirmed by Nothern blot. Cell proliferation assay and soft agar colony formation assay were used to determine the status of the cells proliferation. Cell cycle checkpoints including cyclin E and cyclin D1 were examined by Western blot. Results: The results from cell proliferation assays suggested that down-regulation of LRP16 gene expression is capable of inhibiting MCF-7 breast cancer cell growth and down-regulation of the LRP16 gene expression is able to inhibit anchorage-independent growth of breast cancer cells in soft agar. We also demonstrated that cyclin E and cyclin D1 proteins were much lower in the LRP16-inhibitory cells than in the control cells. Conclusion: These data suggest that LRP16 gene play an important role in MCF-7 cells proliferation by regulating the pathway of the G1/S transition and may function as an important modulator in regulating the process of tumorigenesis in human breast.

  1. Identification and characterization of gene-based SSR markers in date palm (Phoenix dactylifera L.

    Zhao Yongli


    Full Text Available Abstract Background Date palm (Phoenix dactylifera L. is an important tree in the Middle East and North Africa due to the nutritional value of its fruit. Molecular Breeding would accelerate genetic improvement of fruit tree through marker assisted selection. However, the lack of molecular markers in date palm restricts the application of molecular breeding. Results In this study, we analyzed 28,889 EST sequences from the date palm genome database to identify simple-sequence repeats (SSRs and to develop gene-based markers, i.e. expressed sequence tag-SSRs (EST-SSRs. We identified 4,609 ESTs as containing SSRs, among which, trinucleotide motifs (69.7% were the most common, followed by tetranucleotide (10.4% and dinucleotide motifs (9.6%. The motif AG (85.7% was most abundant in dinucleotides, while motifs AGG (26.8%, AAG (19.3%, and AGC (16.1% were most common among trinucleotides. A total of 4,967 primer pairs were designed for EST-SSR markers from the computational data. In a follow up laboratory study, we tested a sample of 20 random selected primer pairs for amplification and polymorphism detection using genomic DNA from date palm cultivars. Nearly one-third of these primer pairs detected DNA polymorphism to differentiate the twelve date palm cultivars used. Functional categorization of EST sequences containing SSRs revealed that 3,108 (67.4% of such ESTs had homology with known proteins. Conclusion Date palm EST sequences exhibits a good resource for developing gene-based markers. These genic markers identified in our study may provide a valuable genetic and genomic tool for further genetic research and varietal development in date palm, such as diversity study, QTL mapping, and molecular breeding.

  2. Association analysis of peroxisome proliferator-activated receptors gamma gene polymorphisms with asprin hypersensitivity in asthmatics

    Oh, Sun-Hee; Park, Se-Min; Park, Jong-Sook; Jang, An-Soo; Lee, Yong-Mok; Uh, Soo-Taek; Kim, Young Hoon; Choi, In-Seon; Kim, Mi-Kyeong; Park, Byeong Lae


    Purpose Peroxisome proliferator-activated receptors (PPARs) are transcriptional factors activated by ligands of the nuclear hormone receptor superfamily. The activation of PPARγ regulates inflammation by downregulating the production of Th2 type cytokines and eosinophil function. In addition, a range of natural substances, including arachidonate pathway metabolites such as 15-hydroxyeicosatetranoic acid (15-HETE), strongly promote PPARG expression. Therefore, genetic variants of the PPARG gene may be associated with the development of aspirin-intolerant asthma (AIA). We investigated the relationship between single nucleotide polymorphism (SNP) of the PPARG gene and AIA. Methods Based on the results of an oral aspirin challenge, asthmatics (n=403) were categorized into two groups: those with a decrease in FEV1 of 15% or greater (AIA) or less than 15% (aspirin-tolerant asthma, ATA). We genotyped two single nucleotide polymorphisms in the PPARG gene from Korean asthmatics and normal controls (n=449): +34C>G (Pro12Ala) and +82466C>T (His449His). Results Logistic regression analysis showed that +82466C>T and haplotype 1 (CC) were associated with the development of aspirin hypersensitivity in asthmatics (P=0.04). The frequency of the rare allele of +82466C>T was significantly higher in AIA patients than in ATA patients in the recessive model [P=0.04, OR=3.97 (1.08-14.53)]. In addition, the frequency of PPARG haplotype 1 was significantly lower in AIA patients than in ATA patients in the dominant model (OR=0.25, P=0.04). Conclusions The +82466C>T polymorphism and haplotype 1 of the PPARG gene may be linked to increased risk for aspirin hypersensitivity in asthma. PMID:20224667

  3. PCNA--a cell proliferation marker in vocal cord cancer. Part II: Recurrence in malignant laryngeal lesions.

    Broich, G; Lavezzi, A M; Biondo, B; Pignataro, L D


    Laryngeal squamous cell carcinoma constitutes the most frequent carcinoma found in the head and neck region. A precise prediction for recurrence potential cannot be done on site, treatment and histologic grading. Since Proliferating Cell Nuclear Antigen (PCNA) and DNA-cytometry have shown a good correlation between premalignant lesions and their progressive potential towards full-fledged carcinoma in the larynx as described in part I of this work, we have analyzed the PCNA index and DNA cytometry in specimen taken from vocal chord carcinomas with a 5-year follow-up, in order to assess its relationship with the presence or absence of tumour progression. 42 cases with (21) and without (2) recurrence have been examined. The DNA-index ranged from 1.01 to 1.43 (mean 1.10) in the group without and from 1.02 to 1.59 (mean 1.38) in the group with recurrent carcinoma (p = 0.002). The PCNA-index ranged from 0.00% to 18.90% (mean 6.97%) in the nonrecurrent group and from 0.00 to 3g.50% (mean 16.35%) in the patients with recurrence (p = 0.001). Both indices also correlated in a highly significant way. From these data emerges a highly significant correlation between the cytometric indices of cell proliferation and PCNA immunostaining. Furthermore the high correction between PCNA and DNA-index is of special interest for single case assessment. High DNA aberration and PCNA-index in vocal chord carcinoma may indicate a higher cellular aggressiveness of the tumour, resulting in a greater overall risk of metastases and local recurrences. Our results support the thesis that the indices of cellular proliferation within some cancers can define subsets of patients of high risk and help in isolating a population in which a more aggressive clinical protocol may be proposed.

  4. Tagging microsatellite marker to a blast resistance gene in the irrigated rice cultivar Cica-8

    Thiago Martins Pinheiro


    Full Text Available The rice cultivar Cica-8 exhibit differential reaction to several pathotypes of Magnaporthe oryzae. The objective of the present investigation was to determine the number of alleles involved in the expression of resistance to leaf blast and identify microsatellite markers linked to these alleles. A cross between cultivar Metica-1 and Cica-8 susceptible and resistant, respectively, to pathotype IB-1 (Py1049 was made to obtain F1, F2, BC1:1 and BC1:2 progenies. Greenhouse tests for leaf blast reaction showed that resistance is controlled by a monogenic dominant gene. For testing microsatellite markers, DNA of both resistant and susceptible parents and F1 and F2 populations was extracted. As expected for single dominant gene the F2 populations segregated at a ratio of 3:1. Of the 11 microsatellite markers tested, one marker RM 7102 was found to be closely linked to the resistant allele at a distance of 2.7 cM, in the cultivar Cica-8 to pathotype IB-1.

  5. Genetic Diversity of Japonica Rice (Oryza sativa L. Based on Markers Corresponding to Starch Synthesizing Genes

    Puji Lestari


    Full Text Available Genes related to starch synthesis and the metabolism contribute to a variety of physicochemical properties that determine the eating/cooking qualities of rice. Our previous study suggested that a set of molecular markers was able to estimate the eating quality of japonica rice. The present study reports the genetic diversity of 22 japonica rice varieties based on markers corresponding to starch synthesizing genes. The mean of the polymorphic information content (PIC: 0.135 value and the diversity index (0.171 indicated a low genetic diversity in these varieties. The phylogenetic tree clearly demonstrated three main clusters: 1 cluster I contained seven varieties with similar physicochemical properties; 2 cluster II only showed a Japanese variety, Koshihikari, and 3 cluster III included the most Korean varieties (14 varieties. This phylogenetic analysis did not completely represent the physicochemical properties differentiation of the japonica varieties, although it did reveal an initial clue to the close relationship between Korean rice and the Japanese and Chinese varieties. Notably, these markers were also able to identify a premium japonica rice. The molecular markers and information concerning the genetic relationship would be useful in improving the japonica rice along with its starch quality of in breeding program.

  6. Elimination of marker genes from transgenic plants using MAT vector systems.

    Ebinuma, Hiroyasu; Sugita, Koichi; Endo, Saori; Matsunaga, Etsuko; Yamada, Keiko


    We have developed an efficient system (Multi-Auto-Transformation [MAT] vectors) for the removal of marker genes and to increase the regeneration frequency of transgenic crops without using antibiotic selection, reducing their possible environmental impact. The MAT vector system is designed to use the oncogenes (ipt, iaaM/H, rol) of Agrobacterium, which control the endogenous levels of plant hormones and the cell responses to plant growth regulators, to differentiate transgenic cells, and to select marker-free transgenic plants. The oncogenes are combined with the site-specific recombination system (R/RS). At transformation, the oncogenes regenerate transgenic plants and then are removed by the R/RS system to generate marker-free transgenic plants. The choice of a promoter for the oncogenes and the recombinase (R) gene, the state of plant materials and the tissue culture conditions greatly affect efficiency of both the regeneration of transgenic plants and the generation of marker-free plants. We have evaluated these conditions in several plant species to increase their generation efficiency. This chapter describes our transformation protocols using MAT vectors.

  7. Identification of Co-Segregating RAPD Marker Linked to Powdery Mildew Resistance Gene Pm 18 in Wheat

    ZHANG Qing-li; GU Feng; LI Tao; GAO Ju-rong; WANG Hong-gang


    The Pm18 gene of wheat confers resistance to the powdery mildew which is oneof the most serious diseases in many regions of the world. In this study, bulked segregant analysis (BSA) was used to develop randomly amplified polymorphic DNA (RAPD) markers linked to Pml8 gene. Three hundred and twenty decamer primers were screened and one of them was identified as RAPD marker (S411600) linked to Pml8. Using the F2 mapping population from the cross Pml8 × Chancellor, the marker S411600 was shown to co-segregate with the gene Pml8. This marker can be conveniently used for marker-assisted selection in wheat breeding programs for the identification or pyramiding of Pml8 with other resistance genes.

  8. SCAR, RAPD and RFLP markers linked to a dominant gene (Are) conferring resistance to anthracnose in common bean.

    Adam-Blondon, A F; Sévignac, M; Bannerot, H; Dron, M


    Anthracnose, caused by the fungusColletotrichum lindemuthianum, is a severe disease of common bean (Phaseolus vulgaris L.) controlled, in Europe, by a single dominant gene,Are. Four pairs of near-isogenic lines (NILs) were constructed, in which theAre gene was introgressed into different genetic backgrounds. These pairs of NILs were used to search for DNA markers linked to the resistance gene. Nine molecular markers, five RAPDs and four RFLPs, were found to discriminate between the resistant and the susceptible members of these NILs. A backcross progeny of 120 individuals was analysed to map these markers in relation to theAre locus. Five out of the nine markers were shown to be linked to theAre gene within a distance of 12.0 cM. The most tightly linked, a RAPD marker, was used to generate a pair of primers that specifically amplify this RAPD (sequence characterized amplified region, SCAR).

  9. Millions of reads, thousands of taxa: microbial community structure and associations analyzed via marker genes.

    Bálint, Miklós; Bahram, Mohammad; Eren, A Murat; Faust, Karoline; Fuhrman, Jed A; Lindahl, Björn; O'Hara, Robert B; Öpik, Maarja; Sogin, Mitchell L; Unterseher, Martin; Tedersoo, Leho


    With high-throughput sequencing (HTS), we are able to explore the hidden world of microscopic organisms to an unpre-cedented level. The fast development of molecular technology and statistical methods means that microbial ecologists must keep their toolkits updated. Here, we review and evaluate some of the more widely adopted and emerging techniques for analysis of diversity and community composition, and the inference of species interactions from co-occurrence data generated by HTS of marker genes. We emphasize the importance of observational biases and statistical properties of the data and methods. The aim of the review is to critically discuss the advantages and disadvantages of established and emerging statistical methods, and to contribute to the integration of HTS-based marker gene data into community ecology.

  10. Hidden histories of gene flow in highland birds revealed with genomic markers.

    Zarza, Eugenia; Faircloth, Brant C; Tsai, Whitney L E; Bryson, Robert W; Klicka, John; McCormack, John E


    Genomic studies are revealing that divergence and speciation are marked by gene flow, but it is not clear whether gene flow has played a prominent role during the generation of biodiversity in species-rich regions of the world where vicariance is assumed to be the principal mode by which new species form. We revisit a well-studied organismal system in the Mexican Highlands, Aphelocoma jays, to test for gene flow among Mexican sierras. Prior results from mitochondrial DNA (mtDNA) largely conformed to the standard model of allopatric divergence, although there was also evidence for more obscure histories of gene flow in a small sample of nuclear markers. We tested for these 'hidden histories' using genomic markers known as ultraconserved elements (UCEs) in concert with phylogenies, clustering algorithms and newer introgression tests specifically designed to detect ancient gene flow (e.g. ABBA/BABA tests). Results based on 4303 UCE loci and 2500 informative SNPs are consistent with varying degrees of gene flow among highland areas. In some cases, gene flow has been extensive and recent (although perhaps not ongoing today), whereas in other cases there is only a trace signature of ancient gene flow among species that diverged as long as 5 million years ago. These results show how a species complex thought to be a model for vicariance can reveal a more reticulate history when a broader portion of the genome is queried. As more organisms are studied with genomic data, we predict that speciation-with-bouts-of-gene-flow will turn out to be a common mode of speciation.

  11. Altruism can proliferate through population viscosity despite high random gene flow.

    Roberto H Schonmann

    Full Text Available The ways in which natural selection can allow the proliferation of cooperative behavior have long been seen as a central problem in evolutionary biology. Most of the literature has focused on interactions between pairs of individuals and on linear public goods games. This emphasis has led to the conclusion that even modest levels of migration would pose a serious problem to the spread of altruism through population viscosity in group structured populations. Here we challenge this conclusion, by analyzing evolution in a framework which allows for complex group interactions and random migration among groups. We conclude that contingent forms of strong altruism that benefits equally all group members, regardless of kinship and without greenbeard effects, can spread when rare under realistic group sizes and levels of migration, due to the assortment of genes resulting only from population viscosity. Our analysis combines group-centric and gene-centric perspectives, allows for arbitrary strength of selection, and leads to extensions of Hamilton's rule for the spread of altruistic alleles, applicable under broad conditions.

  12. KCNE gene expression is dependent on the proliferation and mode of activation of leukocytes.

    Solé, Laura; Vallejo-Gracia, Albert; Roig, Sara R; Serrano-Albarrás, Antonio; Marruecos, Laura; Manils, Joan; Gómez, Diana; Soler, Concepció; Felipe, Antonio


    Voltage-dependent K (+) (Kv) channels are tightly regulated during the immune system response. Leukocytes have a limited repertoire of Kv channels, whose physiological role is under intense investigation. A functional Kv channel is an oligomeric complex composed of pore-forming and ancillary subunits. The KCNE gene family is a novel group of modulatory Kv channel elements in leukocytes. Here, we characterized the gene expression of KCNEs (1-5) in leukocytes and investigated their regulation during leukocyte proliferation and mode of activation. Murine bone-marrow-derived macrophages, human Jurkat T-lymphocytes and human Raji B-cells were analyzed. KCNEs (1-5) are expressed in all leukocytes lineages. Most KCNE mRNAs show cell cycle-dependent regulation and are differentially regulated under specific insults. Our results further suggest a new and yet undefined physiological role for KCNE subunits in the immune system. Putative associations of these ancillary proteins with Kv channels would yield a wide variety of biophysically and pharmacologically distinct channels that fine-tune the immunological response.

  13. The X protein of hepatitis B virus activates hepatoma cell proliferation through repressing melanoma inhibitory activity 2 gene

    Xu, Yilin; Yang, Yang; Cai, Yanyan; Liu, Fang; Liu, Yingle; Zhu, Ying [State Key Laboratory of Virology, College of Life Sciences, and Chinese-French Liver Disease Research Institute at Zhongnan Hospital, Wuhan University, Wuhan 430072 (China); Wu, Jianguo, E-mail: [State Key Laboratory of Virology, College of Life Sciences, and Chinese-French Liver Disease Research Institute at Zhongnan Hospital, Wuhan University, Wuhan 430072 (China)


    Highlights: Black-Right-Pointing-Pointer We demonstrated that HBV represses MIA2 gene expression both invitro and in vivo. Black-Right-Pointing-Pointer The X protein of HBV plays a major role in such regulation. Black-Right-Pointing-Pointer Knock-down of MIA2 in HepG2 cells activates cell growth and proliferation. Black-Right-Pointing-Pointer HBx activates cell proliferation, over-expression of MIA2 impaired such regulation. Black-Right-Pointing-Pointer HBx activates hepatoma cell proliferation through repressing MIA2 expression. -- Abstract: Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer deaths globally. Chronic hepatitis B virus (HBV) infection accounts for over 75% of all HCC cases; however, the molecular pathogenesis of HCC is not well understood. In this study, we found that the expression of the newly identified gene melanoma inhibitory activity 2 (MIA2) was reduced by HBV infection invitro and invivo, and that HBV X protein (HBx) plays a major role in this regulation. Recent studies have revealed that MIA2 is a potential tumor suppressor, and that, in most HCCs, MIA2 expression is down-regulated or lost. We found that the knock-down of MIA2 in HepG2 cells activated cell growth and proliferation, suggesting that MIA2 inhibits HCC cell growth and proliferation. In addition, the over-expression of HBx alone induced cell proliferation, whereas MIA2 over-expression impaired the HBx-mediated induction of proliferation. Taken together, our results suggest that HBx activates hepatoma cell growth and proliferation through repression of the potential tumor suppressor MIA2.

  14. Effects of AFP gene silencing on apoptosis and proliferation of a hepatocellular carcinoma cell line.

    Zhang, Ling; He, Tao; Cui, Hong; Wang, Yunjian; Huang, Changshan; Han, Feng


    Alpha fetoprotein (AFP) is an oncoembryonal protein that is highly expressed in the majority of hepatocellular carcinomas. Previous studies have shown that AFP may be involved in multiple cell growth regulating, differentiating, and immunosuppressive activities. We investigated the effects of AFP gene silencing by siRNA on apoptosis and proliferation of hepatocellular carcinoma cell line EGHC-9901, which highly expresses AFP and may serve as an ideal model for investigation of AFP functions. siRNA expressing plasmid targeting the AFP gene was first established and subsequently transfected into hepatocellular carcinoma cell line EGHC-9901; cells were then divided into three groups: siRNA-afp, transfected with AFP-siRNA; siRNA-beta-actin, transfected with siRNA-beta-actin as the positive group; and vector control, transfected with empty vector as the blank control group. After G418 positive clone selection for a couple of weeks, Western blot and RT (reverse transcription)-PCR assay demonstrated that AFP expression was almost completely inhibited by siRNA-afp, which indicates that siRNA expressing plasmid targeting the AFP gene has been successfully established. Furthermore, MTT (methyl thiazolyl tetrazelium) assay showed that cells transfected with siRNA-afp proliferated at a significantly lower speed than the other two groups and flat plate clone formation assay also witnessed less clones with diameters of more than 75 μm in siRNA-afp immunofluorescence indicating that the apoptosis rate of cells transfected with siRNA-afp was significantly higher than the other two groups. Furthermore, flow cytometry manifested approximately 20% more cells of siRNA-afp within G1 phase than those of the negative group, indicating that inhibition of AFP expression may cause G1 phase arrest. Finally, Western blot and RT-PCR assay demonstrated that siRNA-afp induced a higher expression of caspase-3 than the other two groups whereas there was no difference in expression of caspase-8

  15. Apoptosis, proliferation and p53 gene expression of H. pylori associated gastric epithelial lesions

    Zhong Zhang1; Yuan Yuan; Hua Gao; Ming Dong; Lan Wang; Yue-Hua Gong


    AIM: To study the relationship between Helicobacter pylori (H. Pylori) and gastric carcinoma and its possible pathogenesis by H. Pylori. METHODS: DNEL technique and immunohistochemical technique were used to study the state of apoptosis,proliferation and p53 gene expression. A total of 100 gastric mucosal biopsy specimens, including 20 normal mucosa, 30H. Pylori-negative and 30 H. Pylorf-positive gastric precancerous lesions along with 20 gastric carcinomas were studied. RESULTS: There were several apoptotic cells in the superficial epithelium and a few proliferative cells within the neck of gastric glands, and no p53 protein expression in normal mucosa. In gastric carcinoma, there were few apoptotic cells, while there were a large number of proliferative cells, and expression of p53 protein significantly was increased. In the phase of metaplasia, the apoptotic index (Al, 4.36% ± 1.95%), proliferative index (PI, 19.11% ± 6.79%) and positivity of p53 expression (46.7%) in H. Pylori-positive group were higher than those in normal mucosa (P< 0.01). Al in H. Pylori-positive group was higher than that in H. Pylori-negative group (3.81% ±1.76%), PI in H. Pylori-positive group was higher than that in H. Pylori-negative group (12.25% ±5.63%, P<0.01 ). In the phase of dysplasia, Al (2.31% ± 1.10%) in H. Pylori-positive group was lower (3.05% ± 1.29%) than that in H. Pylori-negative group, but PI (33.89% ± 11.65%)wassignificantly higher(22.09± 8018%, P< 0.01). In phases of metaplasia, dysplasia and gastric cancer in the H. Pylori-positive group, Als had an evidently graduall decreasing trend (P < 0.01 ), while Pis had an evidently gradual increasing trend (P< 0.05 or P< 0.01), and there was also a trend of gradual increase in the expression of p53 gene. CONCLUSION: In the course of the formation of gastric carcinoma, proliferation of gastric mucosa can be greatly increased by H. Pylori, and H. Pylori can induce apoptosis in the phase of metaplasia but in the phase of

  16. SCAR Markers Assisted Selection for a Bentazon Susceptible Lethality Gene (ben) in Rice

    XIANG; Tai-he; YANG; Jian-bo; YANG; Qian-jin; ZHU; Qi-sheng; LI; Li; HUANG; Da-nian


    In progenies resulting from crosses involving rice cultivar Norin 8m susceptible to bentazon as the donor of ben gene, SCARs tightly linked to ben were utilized for selection of ben. The homozygous and heterozygous genotypes with ben could be identified with the SCARs. The molecular markers offer a powerful tool for indirect selection of ben and can accelerate the introgression of ben into current rice cultivars.

  17. A unique mosaic Turner syndrome patient with androgen receptor gene derived marker chromosome.

    Kalkan, Rasime; Özdağ, Nermin; Bundak, Rüveyde; Çirakoğlu, Ayşe; Serakinci, Nedime


    Patients with Turner syndrome are generally characterized by having short stature with no secondary sexual characteristics. Some abnormalities, such as webbed neck, renal malformations (>50%) and cardiac defects (10%) are less common. The intelligence of these patients is considered normal. Non-mosaic monosomy X is observed in approximately 45% of postnatal patients with Turner syndrome and the rest of the patients have structural abnormalities or mosaicism involving 46,X,i(Xq), 45,X/46,XX, 45,X and other variants. The phenotype of 45,X/46,X,+mar individuals varies by the genetic continent and degree of the mosaicism. The gene content of the marker chromosome is the most important when correlating the phenotype with the genotype. Here we present an 11-year-old female who was referred for evaluation of her short stature and learning disabilities. Conventional cytogenetic investigation showed a mosaic 45,X/46,X,+mar karyotype. Fluorescence in situ hybridization showed that the marker chromosome originated from the X chromosome within the androgen receptor (AR) and X-inactive specific transcript (XIST) genes. Therefore, it is possible that aberrant activation of the marker chromosome, compromising the AR and XIST genes, may modify the Turner syndrome phenotype.

  18. Gene polymorphisms in association with emerging cardiovascular risk markers in adult women

    Dowling Nicole F


    Full Text Available Abstract Background Evidence on the associations of emerging cardiovascular disease risk factors/markers with genes may help identify intermediate pathways of disease susceptibility in the general population. This population-based study is aimed to determine the presence of associations between a wide array of genetic variants and emerging cardiovascular risk markers among adult US women. Methods The current analysis was performed among the National Health and Nutrition Examination Survey (NHANES III phase 2 samples of adult women aged 17 years and older (sample size n = 3409. Fourteen candidate genes within ADRB2, ADRB3, CAT, CRP, F2, F5, FGB, ITGB3, MTHFR, NOS3, PON1, PPARG, TLR4, and TNF were examined for associations with emerging cardiovascular risk markers such as serum C-reactive protein, homocysteine, uric acid, and plasma fibrinogen. Linear regression models were performed using SAS-callable SUDAAN 9.0. The covariates included age, race/ethnicity, education, menopausal status, female hormone use, aspirin use, and lifestyle factors. Results In covariate-adjusted models, serum C-reactive protein concentrations were significantly (P value controlling for false-discovery rate ≤ 0.05 associated with polymorphisms in CRP (rs3093058, rs1205, MTHFR (rs1801131, and ADRB3 (rs4994. Serum homocysteine levels were significantly associated with MTHFR (rs1801133. Conclusion The significant associations between certain gene variants with concentration variations in serum C-reactive protein and homocysteine among adult women need to be confirmed in further genetic association studies.

  19. Confirmation of root-knot nematode resistant gene Rmi1 using SSR markers

    Musarrat Ramzan


    Full Text Available Background: The Root Knot Nematode (RKN is a serious economic threat to various cultivated crops worldwide. It is a devastating pest of soybean and responsible to cause severe yield loss in Pakistan. The cultivation of resistant soybean varieties against this pest is the sustainable strategy to manage the heavy loss and increase yield. There is an utmost need to identify RKN resistant varieties of soybean against cultivated in Pakistan. The presented study is an attempt to identify and confirm the presence of resistant gene Rmi1 in soybean. Method: Molecular studies have been done using Simple Sequence Repeat (SSR marker system to identify resistant soybean varieties against Root Knot Nematode (RKN using fifteen (15 indigenous cultivars and four (4 US cultivars. DNA was isolated, purified, quantified and then used to employ various SSR markers. The amplified product is observed using gel documentation system after electrophoresis. Results: Diagnostic SSR markers Satt-358 and Satt-492 have shown the presence of Rmi1 gene in all resistance carrying genotypes. Satt-358 amplified the fragment of 200 bp and Satt-492 generated 232 bp bands in all resistant genotypes. This study confirmed the Rmi gene locus (G248A-1 in all internationally confirmed resistant including six (6 native varieties. Conclusion: These investigations have identified six (6 resistant cultivars revealing the effective and informative sources that can be utilized in breeding programs for the selection of RKN resistance soybean genotypes in Pakistan.

  20. Chromosomal Location of Gene for Earbranching of Barley Natural Mutant "f151" Using SSR Markers

    Feng Zongyun(冯宗云); Zhang Lili; Zhang Yizheng; Ling Hongqing


    f151, a natural earbranching mutant from naked barley landrace, has better structural characteristics of spike and is thought to be a very valuable germplasm to high-yield breeding of barley. Genetic analysis of earbranching trait is carried out in the populations of F1, F2, B1 and B2 which are produced by crossing including reciprocals and backcrossing between f151 and different barley varieties. The results show that earbranching trait of f151 is controlled by one pair of recessive genes without cytoplasm effects. The linkage between the earbranching gene and the SSR marker HVM40 on the short arm of chromosome 4H is found by bulked segregated analysis using SSR markers based on the F2 population of the hybrid "f151×Gateway". It can be inferred from the recombinant value of 0.087 between the gene and HVM40 that this gene is located on 4HS. The gene is temporarily named "mb".

  1. Precision genome editing in plants via gene targeting and piggyBac-mediated marker excision.

    Nishizawa-Yokoi, Ayako; Endo, Masaki; Ohtsuki, Namie; Saika, Hiroaki; Toki, Seiichi


    Precise genome engineering via homologous recombination (HR)-mediated gene targeting (GT) has become an essential tool in molecular breeding as well as in basic plant science. As HR-mediated GT is an extremely rare event, positive-negative selection has been used extensively in flowering plants to isolate cells in which GT has occurred. In order to utilize GT as a methodology for precision mutagenesis, the positive selectable marker gene should be completely eliminated from the GT locus. Here, we introduce targeted point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with subsequent marker excision by piggyBac transposition. Almost all regenerated plants expressing piggyBac transposase contained exclusively targeted point mutations without concomitant re-integration of the transposon, resulting in these progeny showing a herbicide bispyribac sodium (BS)-tolerant phenotype. This approach was also applied successfully to the editing of a microRNA targeting site in the rice cleistogamy 1 gene. Therefore, our approach provides a general strategy for the targeted modification of endogenous genes in plants.

  2. Molecular Markers for Leaf Rust Resistance Gene Lr45 in Wheat Based on AFLP Analysis

    ZHANG Na; YANG Wen-xiang; YAN Hong-fei; LIU Da-qun; CHU Dong; MENG Qing-fang; ZHANG Ting


    Amplified fragment length polymorphism (AFLP) analysis was carried out in Thatcher, near isogenic lines (NILs) carrying different genes conferring resistance against wheat leaf rust, and TcLr45×Thatcher F2 progenies were used to develop markers for Lr45 gene. Sixty AFLP primer combinations were screened and most of them provided clear amplification products, 31 primer combinations displayed polymorphism of TcLr45 in 23 NILs. Two AFLP markers closely linked to the gene Lr45 were acquired: P-AGG/M-GAG261 bp, which was found closely linked to the Lr45 locus at a distance of 0.6 cM on one side, and P-ACA/M-GGT105 bp, which was found at a distance of 1.3 cM on the other side. The specific bands were cloned and subsequently sequenced. The 261-bp fragment produced by P-AGG/M-GAG showed 86% similarity with the sequence of Vulgare Hort Ⅰ gene; the 105-bp fragment produced by P-ACA/M-GGT showed 96% similarity with the phosphatidylserine decarboxylase gene of the Triticum monococcum. Both included an open reading frame (ORF).

  3. Stable reporter cell lines for peroxisome proliferator-activated receptor y (PPARy)-mediated modulation of gene expression

    Gijsbers, L.; Man, H.Y.; Kloet, S.K.; Haan, de L.H.J.; Keijer, J.; Rietjens, I.; Burg, van der B.J.; Aarts, J.M.M.J.G.


    Activation of peroxisome proliferator-activated receptor ¿ (PPAR¿) by ligands is associated with beneficial health effects, including anti-inflammatory and insulin-sensitizing effects. The aim of the current study was to develop luciferase reporter gene assays to enable fast and low-cost measurement

  4. Regulation of deleted in liver cancer-1 gene domains on the proliferation of human colon cancer HT29 cell



    Objective To study the role of deleted in liver cancer-1(DLC-1) gene main domains on the regulation of hu-man colon cancer HT29 cell proliferation. Methods Subcloning recombinant plasmid vectors with Rho GTPase activating protein(RhoGAP),sterile alpha motif(SAM)

  5. Proliferation, behavior, and cytokine gene expression of human umbilical vascular endothelial cells in response to different titanium surfaces.

    An, Na; Schedle, Andreas; Wieland, Marco; Andrukhov, Oleh; Matejka, Michael; Rausch-Fan, Xiaohui


    Success of dental implantation is initially affected by wound healing of both, hard and soft tissues. Endothelial cells (ECs) are involved as crucial cells in the angiogenesis and inflammation process of wound healing. In the present study, proliferation, mobility, cluster formation, and gene expression of angiogenesis-related molecules of human umbilical vascular endothelial cells (HUVECs) were investigated on titanium surfaces with different roughnesses: acid-etched (A), coarse-grit-blasted and acid-etched (SLA) surfaces, as well as on hydrophilic modified modA and modSLA surfaces. Cell behaviors were analyzed by proliferation assay and time-lapse microscopy, gene expression was analyzed by real time PCR. Results showed that cell proliferation, mobility, and cluster formation were highest on modA surfaces compared with all other surfaces. HUVECs moved slowly and exhibited seldom cell aggregation on SLA and modSLA surfaces during the whole observing period of 120 h. The gene expressions of the angiogenesis-related factors von Willebrand factor, thrombomodulin, endothelial cell protein C receptor, and adhesion molecules intercellular adhesion molecule-1 and E-selectin were most enhanced on modSLA surfaces. These results suggest that modA surface is optimal for proliferation and angiogenic behavior of ECs. However, modSLA surface seems to promote ECs to express angiogenesis-related factor genes, which play essential roles in controlling inflammation and revascularization of wound healing.

  6. Association of peroxisome proliferator-activated receptor-gamma gene polymorphisms with the development of asthma.

    Oh, Sun-Hee; Park, Se-Min; Lee, Yoo Hoon; Cha, Ji Yeon; Lee, Ji-Yeon; Shin, Eun Kyong; Park, Jong-Sook; Park, Byeong-Lae; Shin, Hyoung Doo; Park, Choon-Sik


    The peroxisome proliferator-activated receptors (PPAR) are the nuclear hormone receptor superfamily of ligand-activated transcriptional factors. PPAR-gamma (PPARG) activation downregulates production of Th2 type cytokines and eosinophil function. Additionally, treatment with a synthetic PPARG ligand can reduce lung inflammation and IFN-gamma, IL-4, and IL-2 production in experimental allergic asthma. In patients with asthma, PPARG gene expression is known to be associated with the airway inflammatory and remodeling responses. Thus, genetic variants of PPARG may be associated with the development of asthma. We genotyped two single nucleotide polymorphisms on the PPARG gene, +34C>G (Pro12Ala) and +82466C>T (His449His), in Korean subjects (839 subjects with asthma and 449 normal controls). Association analysis using logistic regression analysis showed that +82466C>T and haplotypes 1(CC) and 2(CT) were associated with the development of asthma (p=0.01-0.04). The frequency of PPARG-ht2 was significantly lower in the patients with asthma compared to the normal controls in codominant and dominant models (p=0.01, p(corr)=0.03 and p=0.02, p(corr)=0.03, respectively). Conversely, the frequency of PPARG-ht1 was significantly higher in the patients with asthma compared to the normal controls in the codominant model [p=0.04, OR: 1.27 (1.01-1.6)]. In addition, the rare allele frequency of +82466C>T was significantly lower in patients with asthma in comparison to normal controls in the codominant model (OR: 0.78, p=0.04). Thus, polymorphism of the PPARG gene may be linked to an increased risk of asthma development.

  7. Peroxisome proliferator-activated receptors and atherogenesis: regulators of gene expression in vascular cells.

    Marx, Nikolaus; Duez, Hélène; Fruchart, Jean-Charles; Staels, Bart


    A large body of data gathered over the past couple of years has identified the peroxisome proliferator-activated receptors (PPAR) alpha, gamma, and beta/delta as transcription factors exerting modulatory actions in vascular cells. PPARs, which belong to the nuclear receptor family of ligand-activated transcription factors, were originally described as gene regulators of various metabolic pathways. Although the PPARalpha, gamma, and beta/delta subtypes are approximately 60% to 80% homologous in their ligand- and DNA-binding domains, significant differences in ligand and target gene specificities are observed. PPARalpha is activated by polyunsaturated fatty acids and oxidized derivatives and by lipid-modifying drugs of the fibrate family, including fenofibrate or gemfibrozil. PPARalpha controls expression of genes implicated in lipid metabolism. PPARgamma, in contrast, is a key regulator of glucose homeostasis and adipogenesis. Ligands of PPARgamma include naturally occurring FA derivatives, such as hydroxyoctadecadienoic acids (HODEs), prostaglandin derivatives such as 15-deoxyDelta12,14-prostaglandin J2, and glitazones, insulin-sensitizing drugs presently used to treat patients with type 2 diabetes. Ligands for PPARbeta/delta are polyunsaturated fatty acids, prostaglandins, and synthetic compounds, some of which are presently in clinical development. PPARbeta/delta stimulates fatty acid oxidation predominantly acting in muscle. All PPARs are expressed in vascular cells, where they exhibit antiinflammatory and antiatherogenic properties. In addition, studies in various animal models as well as clinical data suggest that PPARalpha and PPARgamma activators can modulate atherogenesis in vivo. At present, no data are available relating to possible effects of PPARbeta/delta agonists on atherogenesis. Given the widespread use of PPARalpha and PPARgamma agonists in patients at high risk for cardiovascular disease, the understanding of their function in the vasculature is

  8. Multiple endocrine neoplasia type I (MEN1): Identification of informative polymorphic markers and candidate genes

    Taggart, R.T.; Qian, C.; An, Y. [Carolinas Medical Center, Charlotte, NC (United States)] [and others


    Linkage and tumor deletion studies have mapped this autosomal dominant disease to 11q12{yields}13. The MEN1 gene product is suspected to be a growth suppressor or regulator. We report here isolation of polymorphic PCR markers used to narrow the nonrecombinant region and localization of genomic clones encoding expressed sequences within this region. 552 genomic clones were isolated from a radiation hybrid (RH) cell line containing a 5-10 Mb region flanking the gene. We screened the RH cell line with 17 markers flanking the MEN1 region to confirm its integrity. The representation of the markers in the panel of genomic clones derived from the RH cell line indicated a 1- to 4-fold representation of the region. A set of radiation hybrids was used to sublocalized genomic and cDNA clones of interest to 3 regions: centromeric, telomeric and a 1.2 Mb nonrecombinant region. Highly polymorphic PCR markers were developed by hybridization of the clones with tetra- and trinucleotide probes 3 of 7 PCR markers (heterozygosity .46-.92) were nonrecombinant with MEN1. The PCR markers were utilized for definition of the critical region and also proved useful for presymptomatic diagnosis. Genomic clones mapped to the 1.2 Mb nonrecombinant region were used to identify expressed sequences corresponding to 5 different genes. One cDNA clone corresponded to a ubiquitously expressed gene sequence located near PYGM. Two major (3.4, 2.5 kb) and one minor transcript (1.8 kb) were found in pancreas, kidney, brain, lung, heart, skeletal muscle, and liver. DNA analysis matched with 2 anonymous cDNA clones in GenBank. The genomic and cDNA clones were used to screen Southern and Northern blots for MEN1 associated rearrangements before attempting SSCP analysis to detect point mutations. The genomic fragment used to identify the corresponding cDNA clones did not detect alterations in MEN1 patients on Southern blots, however additional fragments were identified in one MEN1 patient with the cDNA clones.

  9. Preliminary screening of differentially expressed genes involved in methyl-CpG-binding protein 2 gene-mediated proliferation in human osteosarcoma cells.

    Meng, Gang; Li, Yi; Lv, YangFan; Dai, Huanzi; Zhang, Xi; Guo, Qiao-Nan


    Methyl-CpG-binding protein 2 (MeCP2) is essential in human brain development and has been linked to several cancer types and neuro-developmental disorders. This study aims to screen the MeCP2 related differentially expressed genes and discover the therapeutic targets for osteosarcoma. CCK8 assay was used to detect the proliferation and SaOS2 and U2OS cells. Apoptosis of cells was detected by flow cytometry analysis that monitored Annexin V-APC/7-DD binding and 7-ADD uptake simultaneously. Denaturing formaldehyde agarose gel electrophoresis was employed to examine the quality of total RNA 18S and 28S units. Gene chip technique was utilized to discover the differentially expressed genes correlated with MeCP2 gene. Differential gene screening criteria were used to screen the changed genes. The gene up-regulation or down-regulation more than 1.5 times was regarded as significant differential expression genes. The CCK8 results indicated that the cell proliferation of MeCP2 silencing cells (LV-MeCP2-RNAi) was significantly decreased compared to non-silenced cells (LV-MeCP2-RNAi-CN) (P genes were screened from a total of 49,395 transcripts. Among the total 107 transcripts, 34 transcripts were up-regulated and 73 transcripts were down-regulated. There were five significant differentially expressed genes, including IGFBP4, HOXC8, LMO4, MDK, and CTGF, which correlated with the MeCP2 gene. The methylation frequency of CpG in IGFBP4 gene could achieve 55%. In conclusion, the differentially expressed IGFBP4, HOXC8, LMO4, MDK, and CTGF genes may be involved in MeCP2 gene-mediated proliferation and apoptosis in osteosarcoma cells.

  10. Gene methylation profiles of normal mucosa, and benign and malignant colorectal tumors identify early onset markers

    Vatn Morten


    Full Text Available Abstract Background Multiple epigenetic and genetic changes have been reported in colorectal tumors, but few of these have clinical impact. This study aims to pinpoint epigenetic markers that can discriminate between non-malignant and malignant tissue from the large bowel, i.e. markers with diagnostic potential. The methylation status of eleven genes (ADAMTS1, CDKN2A, CRABP1, HOXA9, MAL, MGMT, MLH1, NR3C1, PTEN, RUNX3, and SCGB3A1 was determined in 154 tissue samples including normal mucosa, adenomas, and carcinomas of the colorectum. The gene-specific and widespread methylation status among the carcinomas was related to patient gender and age, and microsatellite instability status. Possible CIMP tumors were identified by comparing the methylation profile with microsatellite instability (MSI, BRAF-, KRAS-, and TP53 mutation status. Results The mean number of methylated genes per sample was 0.4 in normal colon mucosa from tumor-free individuals, 1.2 in mucosa from cancerous bowels, 2.2 in adenomas, and 3.9 in carcinomas. Widespread methylation was found in both adenomas and carcinomas. The promoters of ADAMTS1, MAL, and MGMT were frequently methylated in benign samples as well as in malignant tumors, independent of microsatellite instability. In contrast, normal mucosa samples taken from bowels without tumor were rarely methylated for the same genes. Hypermethylated CRABP1, MLH1, NR3C1, RUNX3, and SCGB3A1 were shown to be identifiers of carcinomas with microsatellite instability. In agreement with the CIMP concept, MSI and mutated BRAF were associated with samples harboring hypermethylation of several target genes. Conclusion Methylated ADAMTS1, MGMT, and MAL are suitable as markers for early tumor detection.

  11. Micro- and nano-topography to enhance proliferation and sustain functional markers of donor-derived primary human corneal endothelial cells.

    Muhammad, Rizwan; Peh, Gary S L; Adnan, Khadijah; Law, Jaslyn B K; Mehta, Jodhbir S; Yim, Evelyn K F


    One of the most common indications for corneal transplantation is corneal endothelium dysfunction, which can lead to corneal blindness. Due to a worldwide donor cornea shortage, alternative treatments are needed, but the development of new treatment strategies relies on the successful in vitro culture of primary human corneal endothelial cells (HCECs) because transformed cell lines and animal-derived corneal endothelial cells are not desirable for therapeutic applications. Primary HCECs are non-proliferative in vivo and challenging to expand in vitro while maintaining their characteristic cell morphology and critical markers. Biochemical cues such as growth factors and small molecules have been investigated to enhance the expansion of HCECs with a limited increase in proliferation. In this study, patterned tissue culture polystyrene (TCPS) was shown to significantly enhance the expansion of HCECs. The proliferation of HCECs increased up to 2.9-fold, and the expression amount and localization of cell-cell tight junction protein Zona Occludens-1 (ZO-1) was significantly enhanced when grown on 1 μm TCPS pillars. 250 nm pillars induced an optimal hexagonal morphology of HCEC cells. Furthermore, we demonstrated that the topographical effect on tight-junction expression and cell morphology could be maintained throughout each passage, and was effectively 'remembered' by the cells. Higher amount of tight-junction protein expression was maintained at cell junctions when topographic cues were removed in the successive seeding. This topographic memory suggested topography-exposed/induced cells would maintain the enhanced functional markers, which would be useful in cell-therapy based approaches to enable the in situ endothelial cell monolayer formation upon delivery. The development of patterned TCPS culture platforms could significantly benefit those researching human corneal endothelial cell cultivation for cell therapy, and tissue engineering applications.

  12. Upregulation of metastasis-associated gene 2 promotes cell proliferation and invasion in nasopharyngeal carcinoma

    Wu MH


    Full Text Available Minhua Wu,1,2,* Xiaoxia Ye,2,* Xubin Deng,3,* Yanxia Wu,4 Xiaofang Li,4 Lin Zhang11Department of Histology and Embryology, Southern Medical University, Guangzhou, 2Department of Histology and Embryology, Guangdong Medical University, Zhanjiang, 3Affiliated Cancer Hospital of Guangzhou Medical University, Cancer Center of Guangzhou Medical University, Guangzhou, 4Pathological Diagnosis and Research Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang, People’s Republic of China*These authors contributed equally to this workAims: Metastasis-associated gene 2 (MTA2 is reported to play an important role in tumor progression, but little is known about the role of MTA2 in nasopharyngeal carcinoma (NPC. The aim of the study was to explore the expression and function of MTA2 in NPC.Methods: Expression of MTA2 in NPC tissues and cell lines was detected by immunohistochemistry and Western blotting. Relationship between MTA2 expression and clinicopathological features was analyzed. Stable MTA2-overexpressing and MTA2-siliencing NPC cells were established by transfection with plasmids encoding MTA2 cDNA and lentivirus-mediated short hairpin RNA, respectively. Cell viability was determined by Cell Counting Kit-8 and colony formation assay. Cell migration ability was evaluated by wound healing and transwell invasion assay. The impact of MTA2 knockdown on growth and metastasis of CNE2 cells in vivo was determined by nude mouse xenograft models. Expression of several Akt pathway proteins was detected by Western blotting.Results: MTA2 was upregulated in NPC tissues and three NPC cell lines detected (CNE1, CNE2, and HNE1. MTA2 expression was related to clinical stage and lymph node metastasis of patients with NPC. MTA2 upregulation promoted proliferation and invasion of CNE1 cells, while MTA2 depletion had opposite effects on CNE2 cells. Moreover, MTA2 depletion suppressed growth and metastasis of CNE2 cells in vivo. MTA2 overexpression

  13. A multi-resource data integration approach: Identification of candidate genes regulating cell proliferation during neocortical development

    Cynthia M Vied


    Full Text Available Neurons of the mammalian neocortex are produced by proliferating cells located in the ventricular zone (VZ lining the lateral ventricles. This is a complex and sequential process, requiring precise control of cell cycle progression, fate commitment and differentiation. We have analyzed publicly available databases from mouse and human to identify candidate genes that are potentially involved in regulating early neocortical development and neurogenesis. We used a mouse in situ hybridization dataset (The Allen Institute for Brain Science to identify 13 genes (Cdon, Celsr1, Dbi, E2f5, Eomes, Hmgn2, Neurog2, Notch1, Pcnt, Sox3, Ssrp1, Tead2, Tgif2 with high correlation of expression in the proliferating cells of the VZ of the neocortex at early stages of development (E15.5. We generated a similar human brain network using microarray and RNA-seq data (BrainSpan Atlas and identified 407 genes with high expression in the developing human VZ and subventricular zone (SVZ at 8-9 post-conception weeks. Seven of the human genes were also present in the mouse VZ network. The human and mouse networks were extended using available genetic and proteomic datasets through GeneMANIA. A gene ontology search of the mouse and human networks indicated that many of the genes are involved in the cell cycle, DNA replication, mitosis and transcriptional regulation. The reported involvement of Cdon, Celsr1, Dbi, Eomes, Neurog2, Notch1, Pcnt, Sox3, Tead2 and Tgif2 in neural development or diseases resulting from the disruption of neurogenesis validates these candidate genes. Taken together, our knowledge-based discovery method has validated the involvement of many genes already known to be involved in neocortical development and extended the potential number of genes by 100's, many of which are involved in functions related to cell proliferation but others of which are potential candidates for involvement in the regulation of neocortical development.

  14. Genetic variation in the peroxisome proliferator-activated receptor (PPAR) and peroxisome proliferator-activated receptor gamma co-activator 1 (PGC1) gene families and type 2 diabetes.

    Villegas, Raquel; Williams, Scott M; Gao, Yu-Tang; Long, Jirong; Shi, Jiajun; Cai, Hui; Li, Honglan; Chen, Ching-Chu; Tai, E Shyong; Hu, Frank; Cai, Qiuyin; Zheng, Wei; Shu, Xiao-Ou


    We used a two-stage study design to evaluate whether variations in the peroxisome proliferator-activated receptors (PPAR) and the PPAR gamma co-activator 1 (PGC1) gene families (PPARA, PPARG, PPARD, PPARGC1A, and PPARGC1B) are associated with type 2 diabetes (T2D) risk. Stage I used data from a genome-wide association study (GWAS) from Shanghai, China (1019 T2D cases and 1709 controls) and from a meta-analysis of data from the Asian Genetic Epidemiology Network for T2D (AGEN-T2D). Criteria for selection of single nucleotide polymorphisms (SNPs) for stage II were: (1) P < 0.05 in single marker analysis in Shanghai GWAS and P < 0.05 in the meta-analysis or (2) P < 10(-3) in the meta-analysis alone and (3) minor allele frequency ≥ 0.10. Nine SNPs from the PGC1 family were assessed in stage II (an independent set of middle-aged men and women from Shanghai with 1700 T2D cases and 1647 controls). One SNP in PPARGC1B, rs251464, was replicated in stage II (OR = 0.87; 95% CI: 0.77-0.99). Gene-body mass index (BMI) and gene-exercise interactions and T2D risk were evaluated in a combined dataset (Shanghai GWAS and stage II data: 2719 cases and 3356 controls). One SNP in PPARGC1A, rs12640088, had a significant interaction with BMI. No interactions between the PPARGC1B gene and BMI or exercise were observed. © 2013 John Wiley & Sons Ltd/University College London.

  15. Tagging the gene Wbph2 in ARC 10239 resistant to the whitebacked planthopper Sogatella furcifera by using RFLP markers


    @@Gene tagging is the base of marker-assisted breeding for insect resistance in rice. Five genes (Wbph1, Wbph2, Wbph3, Wbph4, and Wbph5) were identified to be responsible for the resistance to the whitebacked planthopper. The gene Wbph2 in ARC 10239 was clarified as a dominant resistant gene to S.furcifera. In present study, ARC 10239 and susceptible Minghui 63 were selected as parents to make a cross for gene tagging.

  16. [Inhibitory effects of tumor suppressor gene PTEN on proliferation and metastasis of breast cancer ZR-75-1 cells].

    Lin, Guan-Ping; Li, Xiang-Yong; Huang, Jin-Wen; Xiong, Liang; Zhou, Ke-Yuan


    Tumor suppressor gene PTEN could not only inhibit the proliferation of cancer cells, but also inhibit their metastasis. However, the mechanism is still unclear. This study was to investigate the effects of PTEN gene on the proliferation and metastasis of human breast cancer ZR-75-1 cells, and explore the mechanisms. Wild-type PTEN (wt-PTEN) plasmid and phosphatase-defective PTEN (G129R-PTEN) plasmid were transfected into ZR-75-1 cells by liposome, respectively. Cell proliferation was detected by MTT assay. Transfected cells were selected by puromycin. The expression of PTEN protein was detected by Western blot. Cell adhesion and invasion were tested by adhesion test and invasion test. The proliferation inhibition rate was significantly higher in wt-PTEN-transfected ZR-75-1 cells than in untransfected cells and G129R-PTEN-transfected cells (42.7% vs. 0% and 2.7%, P0.05). The proliferation inhibition of ZR-75-1 cells was enhanced along with the increase of culture time and concentration of wt-PTEN. wt-PTEN also induced cell apoptosis. PTEN protein was expressed efficiently in the cells transfected with either wt-PTEN or G129R-PTEN. The inhibition rates of adhesion and invasion were significantly higher in wt-PTEN-transfected cells than in G129R-PTEN-transfected cells (65.7% vs. 8.8%, 70.4% vs. 6.9%, PZR-75-1 cells.

  17. Iron homeostasis in Arabidopsis thaliana: transcriptomic analyses reveal novel FIT-regulated genes, iron deficiency marker genes and functional gene networks.

    Mai, Hans-Jörg; Pateyron, Stéphanie; Bauer, Petra


    FIT (FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR) is the central regulator of iron uptake in Arabidopsis thaliana roots. We performed transcriptome analyses of six day-old seedlings and roots of six week-old plants using wild type, a fit knock-out mutant and a FIT over-expression line grown under iron-sufficient or iron-deficient conditions. We compared genes regulated in a FIT-dependent manner depending on the developmental stage of the plants. We assembled a high likelihood dataset which we used to perform co-expression and functional analysis of the most stably iron deficiency-induced genes. 448 genes were found FIT-regulated. Out of these, 34 genes were robustly FIT-regulated in root and seedling samples and included 13 novel FIT-dependent genes. Three hundred thirty-one genes showed differential regulation in response to the presence and absence of FIT only in the root samples, while this was the case for 83 genes in the seedling samples. We assembled a virtual dataset of iron-regulated genes based on a total of 14 transcriptomic analyses of iron-deficient and iron-sufficient wild-type plants to pinpoint the best marker genes for iron deficiency and analyzed this dataset in depth. Co-expression analysis of this dataset revealed 13 distinct regulons part of which predominantly contained functionally related genes. We could enlarge the list of FIT-dependent genes and discriminate between genes that are robustly FIT-regulated in roots and seedlings or only in one of those. FIT-regulated genes were mostly induced, few of them were repressed by FIT. With the analysis of a virtual dataset we could filter out and pinpoint new candidates among the most reliable marker genes for iron deficiency. Moreover, co-expression and functional analysis of this virtual dataset revealed iron deficiency-induced and functionally distinct regulons.

  18. Tumor endothelial marker 5 expression in endothelial cells during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of cell proliferation

    Vallon, Mario, E-mail: [Nuklearmedizinische Klinik und Poliklinik, Technische Universitaet Muenchen, Ismaninger Strasse 22, 81675 Munich (Germany); Rohde, Franziska; Janssen, Klaus-Peter [Chirurgische Klinik und Poliklinik, Technische Universitaet Muenchen, Munich (Germany); Essler, Markus [Nuklearmedizinische Klinik und Poliklinik, Technische Universitaet Muenchen, Ismaninger Strasse 22, 81675 Munich (Germany)


    Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile, an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.

  19. Single-Copy Genes as Molecular Markers for Phylogenomic Studies in Seed Plants.

    Li, Zhen; De La Torre, Amanda R; Sterck, Lieven; Cánovas, Francisco M; Avila, Concepción; Merino, Irene; Cabezas, José Antonio; Cervera, María Teresa; Ingvarsson, Pär K; Van de Peer, Yves


    Phylogenetic relationships among seed plant taxa, especially within the gymnosperms, remain contested. In contrast to angiosperms, for which several genomic, transcriptomic and phylogenetic resources are available, there are few, if any, molecular markers that allow broad comparisons among gymnosperm species. With few gymnosperm genomes available, recently obtained transcriptomes in gymnosperms are a great addition to identifying single-copy gene families as molecular markers for phylogenomic analysis in seed plants. Taking advantage of an increasing number of available genomes and transcriptomes, we identified single-copy genes in a broad collection of seed plants and used these to infer phylogenetic relationships between major seed plant taxa. This study aims at extending the current phylogenetic toolkit for seed plants, assessing its ability for resolving seed plant phylogeny, and discussing potential factors affecting phylogenetic reconstruction. In total, we identified 3,072 single-copy genes in 31 gymnosperms and 2,156 single-copy genes in 34 angiosperms. All studied seed plants shared 1,469 single-copy genes, which are generally involved in functions like DNA metabolism, cell cycle, and photosynthesis. A selected set of 106 single-copy genes provided good resolution for the seed plant phylogeny except for gnetophytes. Although some of our analyses support a sister relationship between gnetophytes and other gymnosperms, phylogenetic trees from concatenated alignments without 3rd codon positions and amino acid alignments under the CAT + GTR model, support gnetophytes as a sister group to Pinaceae. Our phylogenomic analyses demonstrate that, in general, single-copy genes can uncover both recent and deep divergences of seed plant phylogeny. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  20. Development of co-dominant SCAR markers linked to resistant gene against the Fusarium oxysporum f. sp. radicis-lycopersici.

    Mutlu, Nedim; Demirelli, Aylin; Ilbi, Hülya; Ikten, Cengiz


    We developed highly reliable co-dominant SCAR markers linked to the Frl gene. FORL testing is difficult. The marker is expected to be quickly adapted for MAS by tomato breeders. Fusarium oxysporum f. sp. radicis-lycopersici causes Fusarium crown and root rot (FCR), an economically important soil-borne disease of tomato. The resistance against FCR is conferred by a single dominant gene (Frl) located on chromosome 9. The aim of this study was to develop molecular markers linked to the Frl gene for use in marker-assisted breeding (MAS) programs. The FCR-resistant 'Fla. 7781' and susceptible 'B560' lines were crossed, and F1 was both selfed and backcrossed to 'B560' to generate segregating F2 and BC1 populations. The two conserved set II (COSII) markers were found linked to the Frl gene, one co-segregated with FCR resistance in both F2 and BC1 populations and the other was 8.5 cM away. Both COSII markers were converted into co-dominant SCAR markers. SCARFrl marker produced a 950 and a 1000 bp fragments for resistant and susceptible alleles, respectively. The linkage of SCARFrl marker was confirmed in BC2F3 populations developed by backcrossing the resistant 'Fla. 7781' to five different susceptible lines. The SCARFrl marker has been in use in the tomato breeding programs in BATEM, Antalya, Turkey, since 2012 and has proved highly reliable. The SCARFrl marker is expected to aid in the development of FCR-resistant lines via marker-assisted selection (MAS).

  1. Stratifying melanoma and breast cancer TCGA datasets on the basis of the CNV of transcription factor binding sites common to proliferation- and apoptosis-effector genes.

    Mauro, James A; Yavorski, John M; Blanck, George


    Transcription factors that activate both proliferation- and apoptosis-effector genes, along with a number of related observations, have led to a proposal for a feed forward mechanism of activating the two gene classes, whereby a certain concentration of a transcription factor activates the proliferation-effector genes and a higher concentration of the transcription factor activates the apoptosis-effector genes. We reasoned that this paradigm of regulation could lead to, in the cancer setting, a selection for relatively reduced copy numbers of apoptosis-effector gene, transcription factor binding sites (TFBS). Thus, the aim of this investigation was to examine the DNA sequencing read depths of TFBS for a set of proliferation- and apoptosis-effector genes, normalized to the read depths found in matching blood samples, as provided by the cancer genome atlas (TCGA); and thereby document copy number differences among these TFBS. We determined that the melanoma and breast cancer, TCGA datasets could be divided into three categories: (i) no detectable copy number variation for the proliferation- and apoptosis-effector, shared TFBS; (ii) a relative increase in the copy number of proliferation-effector gene TFBS, compared with the copy number of the apoptosis-effector gene TFBS; and (iii) a relative decrease in the number of proliferation-effector gene TFBS. Thus, we conclude that changes in the relative copies of the shared TFBS, for proliferation- and apoptosis-effector genes, have the potential of impacting tumor cell proliferative and apoptotic capacities.

  2. Plasmid selection in Escherichia coli using an endogenous essential gene marker

    Good Liam


    Full Text Available Abstract Background Antibiotic resistance genes are widely used for selection of recombinant bacteria, but their use risks contributing to the spread of antibiotic resistance. In particular, the practice is inappropriate for some intrinsically resistant bacteria and in vaccine production, and costly for industrial scale production. Non-antibiotic systems are available, but require mutant host strains, defined media or expensive reagents. An unexplored concept is over-expression of a host essential gene to enable selection in the presence of a chemical inhibitor of the gene product. To test this idea in E. coli, we used the growth essential target gene fabI as the plasmid-borne marker and the biocide triclosan as the selective agent. Results The new cloning vector, pFab, enabled selection by triclosan at 1 μM. Interestingly, pFab out-performed the parent pUC19-ampicillin system in cell growth, plasmid stability and plasmid yield. Also, pFab was toxic to host cells in a way that was reversed by triclosan. Therefore, pFab and triclosan are toxic when used alone but in combination they enhance growth and plasmid production through a gene-inhibitor interaction. Conclusion The fabI-triclosan model system provides an alternative plasmid selection method based on essential gene over-expression, without the use of antibiotic-resistance genes and conventional antibiotics.

  3. Effects of PCBs and PBDEs on thyroid hormone, lymphocyte proliferation, hematology and kidney injury markers in residents of an e-waste dismantling area in Zhejiang, China.

    Xu, Peiwei; Lou, Xiaoming; Ding, Gangqiang; Shen, Haitao; Wu, Lizhi; Chen, Zhijian; Han, Jianlong; Wang, Xiaofeng


    Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are two typical categories of contaminants released from e-waste dismantling environments. In China, the body burdens of PCBs and PBDEs are associated with abnormal thyroid hormones in populations from e-waste dismantling sites, but the results are limited and contradictory. In this study, we measured the serum levels of PCBs and PBDEs and the thyroid hormone free triiodothyronine (FT3), free thyroxine (FT4) and thyroid-stimulating hormone (TSH) in 40 residents in an e-waste dismantling area and in 15 residents in a control area. Additionally, we also measured some lymphocyte proliferation indexes, hematologic parameters and kidney injury markers, including white blood cells, neutrophils, monocytes, lymphocytes, hemoglobin, platelets, serum creatinine and beta 2-microglobulin (β2-MG). The results indicated that the mean level of ΣPCBs in the exposure group was significantly higher than that in the control group (964.39 and 67.98 ng g(-1), p0.05). We determined that serum levels of FT3, FT4, monocytes and lymphocytes were significantly lower, whereas the levels of neutrophils, hemoglobin, platelets and serum creatinine were significantly higher in the exposed group (pe-waste dismantling environment may increase the body burdens of PCBs and the specific PBDEs congeners in native residents and that the contaminants released from e-waste may contribute to abnormal changes in body levels of thyroid hormone, hematology and kidney injury markers.

  4. Characterization of RAPD Markers, and the RFLP Marker Linked to Powdery Mildew Resistance Gene Derived from Different Accessions of H. villosa

    LI Hui; CHEN Xiao; SHI Ai-nong; KONG Fan-jing; S Leath; J P Murphy; JIA Xu


    The analysis was carried out on performance of the resistance gene from Haynaldia villosa accession of the former Soviet Union to different isolates of Bluemerie graminis. Polymorphisms were revealed between 6D/6V substitution line Pm930640and its pedigree parents using five RAPD markers of OPAN031700, OPAI017oo, OPAL03750, OPAD07480 and OPAG1558oscreened out from 120 random 10-mers primers. Three RAPD markers of OPAN03, OPAI01 and OPAL03 were linked with the resistance gene by analysis of F2 population of Chancellor×Pm930640. Analysis of 29 wheat lines including part of lines conferring the known genes from Pm1 to Pm20 respectively, lines conferring resistance gene from two H. villosaaccessions and the related wheat parents, were analyzed and the results showed that these markers not only linked to thegene resistant to powdery mildew from H. villosa, but also detected different genetic backgrounds. OPAL03750 can beused as the marker to distinguish the different resistant lines from two H. villosa accessions because it was only observedin the materials from H. villosa of the former Soviet Union. RFLP analysis also showed the polymorphisms between twoH. villosa accessions and their derived resistant lines.

  5. Herpesvirus saimiri-mediated delivery of the adenomatous polyposis coli tumour suppressor gene reduces proliferation of colorectal cancer cells.

    Macnab, Stuart A; Turrell, Susan J; Carr, Ian M; Markham, Alex F; Coletta, P Louise; Whitehouse, Adrian


    Colorectal cancer (CRC) is a major cause of cancer-related mortality. A contributing factor to the progression of this disease is sporadic or hereditary mutation of the adenomatous polyposis coli (APC) gene, a negative regulator of the Wnt signalling pathway. Inherited mutations in APC cause the disorder familial adenomatous polyposis (FAP), which leads to CRC development in early adulthood. However, the gene is also disrupted in some 60% of sporadic cancers. Restoration of functional APC may slow the growth of CRC by negatively regulating proliferation-associated genes such as c-myc. Therefore, we have cloned the cDNA of the APC tumour suppressor gene into a replication competent Herpesvirus saimiri (HVS)-based vector to assess APC gene delivery in SW480 and SW620 CRC cell lines. Our results demonstrate that full length APC protein was efficiently expressed from the HVS vector and that transgene expression inhibited proliferation of both the SW480 and the metastatic SW620 cancer cell lines. Moreover, a sustained effect could be observed for at least 8 weeks after initial infection in SW480 cells. In addition, monolayer wounding assays showed a marked reduction in proliferation and migration in HVS-GFP-APC infected cells. We believe that this is the first instance of infectious delivery and APC cDNA expression from a virus-based vector.

  6. Screening of Genes Specifically Expressed in Males of Fenneropenaeus chinensis and Their Potential as Sex Markers

    Shihao Li


    Full Text Available The androgenic gland (AG, playing an important role in sex differentiation of male crustacean, is a target candidate to understand the mechanism of male development and to mine male-specific sex markers. An SSH library (designated as male reproduction-related tissues—SSH library, MRT-SSH library for short was constructed using cDNA from tissues located at the basal part of the 5th pereiopods, including AG and part of spermatophore sac, as tester, and the cDNA from the basal part of the 4th pereiopods of these male shrimp as driver. 402 ESTs from the SSH library were sequenced and assembled into 48 contigs and 104 singlets. Twelve contigs and 14 singlets were identified as known genes. The proteins encoded by the identified genes were categorized, according to their proposed functions, into neuropeptide hormone and hormone transporter, RNA posttranscriptional regulation, translation, cell growth and death, metabolism, genetic information processing, signal transduction/transport, or immunity-related proteins. Eleven highly expressed contigs in the SSH library were selected for validation of the MRT-SSH library and screening sex markers of shrimp. One contig, specifically expressed in male shrimp, had a potential to be developed as a transcriptomic sex marker in shrimp.

  7. Amelogenin is phagocytized and induces changes in integrin configuration, gene expression and proliferation of cultured normal human dermal fibroblasts

    Almqvist, Sofia; Werthén, Maria; Johansson, Anna


    or down-regulation of genes, of which most are involved in cellular growth, migration and differentiation. The effect of amelogenin was exemplified by increased proliferation over 7 days. In conclusion, the beneficial effects of amelogenin on wound healing are possibly conducted by stimulating fibroblast......Fibroblasts are central in wound healing by expressing important mediators and producing and remodelling extracellular matrix (ECM) components. This study aimed at elucidating possible mechanisms of action of the ECM protein amelogenin on normal human dermal fibroblasts (NHDF). Amelogenin at 100...... signalling, proliferation and migration via integrin interactions. It is hypothesized that amelogenin stimulates wound healing by providing connective tissue cells with a temporary extracellular matrix....

  8. Identification of cold-responsive genes in energycane for their use in genetic diversity analysis and future functional marker development

    Breeding for cold tolerance in sugarcane will allow its cultivation as a dedicated biomass crop in cold environments. Development of functional markers to facilitate marker-assisted breeding requires identification of cold stress tolerance genes. Using suppression subtractive hybridization, 465 cold...

  9. Hypermethylated MAL gene – a silent marker of early colon tumorigenesis

    Kallioniemi Anne


    Full Text Available Abstract Background Tumor-derived aberrantly methylated DNA might serve as diagnostic biomarkers for cancer, but so far, few such markers have been identified. The aim of the present study was to investigate the potential of the MAL (T-cell differentiation protein gene as an early epigenetic diagnostic marker for colorectal tumors. Methods Using methylation-specific polymerase chain reaction (MSP the promoter methylation status of MAL was analyzed in 218 samples, including normal mucosa (n = 44, colorectal adenomas (n = 63, carcinomas (n = 65, and various cancer cell lines (n = 46. Direct bisulphite sequencing was performed to confirm the MSP results. MAL gene expression was investigated with real time quantitative analyses before and after epigenetic drug treatment. Immunohistochemical analysis of MAL was done using normal colon mucosa samples (n = 5 and a tissue microarray with 292 colorectal tumors. Results Bisulphite sequencing revealed that the methylation was unequally distributed within the MAL promoter and by MSP analysis a region close to the transcription start point was shown to be hypermethylated in the majority of colorectal carcinomas (49/61, 80% as well as in adenomas (45/63, 71%. In contrast, only a minority of the normal mucosa samples displayed hypermethylation (1/23, 4%. The hypermethylation of MAL was significantly associated with reduced or lost gene expression in in vitro models. Furthermore, removal of the methylation re-induced gene expression in colon cancer cell lines. Finally, MAL protein was expressed in epithelial cells of normal colon mucosa, but not in the malignant cells of the same type. Conclusion Promoter hypermethylation of MAL was present in the vast majority of benign and malignant colorectal tumors, and only rarely in normal mucosa, which makes it suitable as a diagnostic marker for early colorectal tumorigenesis.

  10. Global gene expression profiling reveals SPINK1 as a potential hepatocellular carcinoma marker.

    Aileen Marshall

    Full Text Available BACKGROUND: Liver cirrhosis is the most important risk factor for hepatocellular carcinoma (HCC but the role of liver disease aetiology in cancer development remains under-explored. We investigated global gene expression profiles from HCC arising in different liver diseases to test whether HCC development is driven by expression of common or different genes, which could provide new diagnostic markers or therapeutic targets. METHODOLOGY AND PRINCIPAL FINDINGS: Global gene expression profiling was performed for 4 normal (control livers as well as 8 background liver and 7 HCC from 3 patients with hereditary haemochromatosis (HH undergoing surgery. In order to investigate different disease phenotypes causing HCC, the data were compared with public microarray repositories for gene expression in normal liver, hepatitis C virus (HCV cirrhosis, HCV-related HCC (HCV-HCC, hepatitis B virus (HBV cirrhosis and HBV-related HCC (HBV-HCC. Principal component analysis and differential gene expression analysis were carried out using R Bioconductor. Liver disease-specific and shared gene lists were created and genes identified as highly expressed in hereditary haemochromatosis HCC (HH-HCC were validated using quantitative RT-PCR. Selected genes were investigated further using immunohistochemistry in 86 HCC arising in liver disorders with varied aetiology. Using a 2-fold cut-off, 9 genes were highly expressed in all HCC, 11 in HH-HCC, 270 in HBV-HCC and 9 in HCV-HCC. Six genes identified by microarray as highly expressed in HH-HCC were confirmed by RT qPCR. Serine peptidase inhibitor, Kazal type 1 (SPINK1 mRNA was very highly expressed in HH-HCC (median fold change 2291, p = 0.0072 and was detected by immunohistochemistry in 91% of HH-HCC, 0% of HH-related cirrhotic or dysplastic nodules and 79% of mixed-aetiology HCC. CONCLUSION: HCC, arising from diverse backgrounds, uniformly over-express a small set of genes. SPINK1, a secretory trypsin inhibitor


    N. A. Gorban


    Full Text Available Renal cell carcinoma (RCC is a heterogeneous disease in which the patients survive for months to years. At the present time the prognostic models have no sufficient information or exact prognostic value. Cell proliferation and apoptosis play a key role in cell cycle regulation; and impairment in these processes is commonly detected in different human tumors. The investigation enrolled 76 patients (49 men, 27 women aged 32 to 73 years (mean age 56 ± 7.6 years diagnosed with RCC. The follow-up was 8 to 116 months (mean 36.5 months. All the patients underwent nephrectomy; antibodies against р53, Bcl-2, and Ki-67 were investigated by immunohistochemistry. The expression of p53 and none or reduced expression of Bcl-2 are poor prognostic factors and associated with the metastatic potential of a tumor and with low relapse-free survival. High Ki-67 levels are a risk factor for metastases. A combination of p53 expression and high proliferative activity reflects the aggressive potential of a tumor and suggests the high risk of metastases just at the disease diagnosis and early tumor dissemination. 

  12. Use of the pyrG gene as a food-grade selection marker in Monascus.

    Wang, Bo-hua; Xu, Yang; Li, Yan-ping


    Ma-pyrG was cloned from Monascus aurantiacus AS3.4384 using degenerate PCR with primers designed with an algorithm called CODEHOP, and its complete sequence was obtained by a PCR-based strategy for screening a Monascus fosmid library. Ma-pyrG encodes orotidine-5'-phosphate decarboxylase (OMPdecase), a 283-aminoacid protein with 81% sequence identity to that from Aspergillus flavus NRRL 3357. A pyrG mutant strain from M. aurantiacus AS3.4384, named UM28, was isolated by resistance to 5-fluoroorotic acid after UV mutagenesis. Sequence analysis of this mutated gene revealed that it contained a point mutation at nucleotide position +220. Plasmid pGFP-pyrG, bearing the green fluorescent protein gene (GFP) as a model gene and Ma-pyrG as a selection marker, were constructed. pGFP-pyrG were successfully transformed into UM28 by using the PEG method.

  13. Development of Simple Functional Markers for Low Glutelin Content Gene 1 (Lgc1) in Rice (Oryza sativa)

    CHEN Tao; TIAN Meng-xiang; ZHANG Ya-dong; ZHU Zhen; ZHAO Ling; ZHAO Qing-yong; LIN Jing; ZHOU Li-hui; WANG Cai-lin


    Rice with low glutelin content is suitable as functional food for patients affected by kidney failure. Low glutelin- content gene Lgc1 in rice has a 3.5-kb deletion between two highly similar glutelin genes GluB4 and GluB5, which locates on the short arm of chromosome 2. To improve the selection efficiency in low glutelin-content rice breeding, two molecular markers designated as InDel-Lgc1-1 and InDel-Lgc1-2 were developed to detect the low glutelin-content gene Lgc1. A double PCR detection indicated that combined use of the two markers could easily distinguish the genotypes of Lgc1 from different rice varieties. Therefore, as a simple and low-cost technique, the molecular marker could be widely used to identify different varieties with Lgc1 gene and applied in marker-assisted selection of low glutelin-content rice.

  14. Prognostic Value of Proliferation Markers: Immunohistochemical Ki-67 Expression and Cytometric S-Phase Fraction of Women with Breast Cancer in Libya

    Eramah Ermiah, Abdelbaset Buhmeida, Fathi Abdalla, Ben Romdhane Khaled, Nada Salem, Seppo Pyrhönen, Yrjö Collan


    Full Text Available Background: We evaluated the association of the immunohistochemical Ki-67 expression, and S-phase fraction with clinicopathological variables and patient outcome.Patients and methods: Histological samples from 100 primary Libyan breast carcinoma patients were retrospectively studied with monoclonal antibody to Ki-67. S-phase fraction was determined by DNA image cytometry.Results: The median Ki-67 percentage for all tumors was 27.5%, ranging from 1 to 80% and the median S-phase fraction (SPF was 11%, ranging from 0 to 62 %. Tumors with high Ki-67 expression were found in 76% of patients and with high SPF values in 56%. Ki-67 expression was more frequent in tumors with high SPF than low SPF. High Ki-67 and high SPF were associated with advanced stages, poor differentiation of tumors, positive lymph nodes, and distant metastasis. The Ki-67 was associated with hormone receptor negative tumors. The SPF was higher in young patients (<50 years than in older patients. In the overall population (median follow-up 49 months, patients with high Ki-67 and high SPF had shorter survival time and predicted recurrence than patients with low Ki-67 and low SPF. In a Cox multivariate analysis, high SPF (p= 0.007, hormonal status (p= 0.001 and clinical stage (p=0.005 were independent predictors of disease-specific survival. The Ki-67 (p=0.065 in borderline significance proved to be independent predictor of disease-free survival. The SPF showed more statistically significance with a high grade of malignancy and survival time than Ki-67.Conclusions: The SPF value is useful cell proliferation marker to assess tumor prognosis. These markers may reflect the aggressive behavior of Libyan breast cancer and predict of the recurrence. It is therefore important to take these markers into consideration to select a high risk subgroup of the patients for intensive treatment.

  15. Importance of introns in the growth regulation of mRNA levels of the proliferating cell nuclear antigen gene.


    The steady-state mRNA levels of the proliferating cell nuclear antigen (PCNA) gene are growth regulated. We have begun to identify the elements in the human PCNA gene that participate in its growth regulation by transfecting appropriate constructs in BALB/c3T3 cells. The results can be summarized as follows. (i) The 400 base pairs of the 5'-flanking sequence of the human PCNA gene upstream of the preferred cap site are sufficient for directing expression of a heterologous cDNA (S. Travali, D....

  16. Hypermethylation of the COX-2 gene is a potential prognostic marker for cervical cancer.

    Jo, Hoenil; Kang, Sokbom; Kim, Jae W; Kang, Gyeong H; Park, Noh H; Song, Yong S; Park, Sang Y; Kang, Soon B; Lee, Hyo P


    The aim of the present study was to evaluate the DNA hypermethylation profiles of 14 genes known to be associated with tumor behavior and their clinical significance in cervical cancer. The clinical features of 82 patients with stage IB cervical cancer were analyzed in terms of DNA hypermethylation of 14 genes (hMLH1, p16, COX-2, CDH1, APC, DAPK, MGMT, p14, RASSF1A, RUNX3, TIMP3, FHIT, THBS1, and HLTF). Of 14 genes investigated, only hypermethylation of COX-2 showed significant association with poor disease-free survival (P = 0.001). To further investigate an alteration in COX-2 expression by DNA hypermethylation, immunohistochemistry for COX-2 protein was performed in the cervical cancer tissues. We found no significant association between hypermethylation and expression patterns of the COX-2 gene. The present results suggest that DNA hypermethylation of the COX-2 gene may be a potential prognostic marker in early stage cervical cancer, the underlying mechanism of which is independent of gene silencing.

  17. Molecular Cloning, Expression Profiling, and Marker Validation of the Chicken Myoz3 Gene

    Maosen Ye


    Full Text Available Myozenin3 (Myoz3 has been reported to bind multiple Z-disc proteins and hence play a key role in signal transduction and muscle fiber type differentiation. The purpose of current study is to better understand the basic characteristics of Myoz3. Firstly, we cloned the ORF (open reading frame of the Myoz3 gene. AA (amino acid sequence analysis revealed that the Myoz3 gene encodes a 26 kDa protein which have 97% identities with that of turkey. Expression profiling showed that Myoz3 mRNA is mainly expressed in leg muscle and breast muscle. Furthermore, we investigated Myoz3 gene polymorphisms in two broiler breeds, the Yellow Bantam (YB and the Avian. Five SNPs (single nucleotide polymorphisms were identified in the YB breed and 3 were identified in the Avian breed. Genotypes and haplotype were constructed and their associations with carcass traits were analyzed. In the YB breed, c.516 C>T had a strong effect on both shank bone length and the L⁎ value of breast muscle, and the H1H3 diplotype had the highest FC compared to other diplotypes. The markers identified in this study may serve as useful targets for the marker-assisted selection (MAS of growth and meat quality traits in chickens.

  18. Identification of ecotype-specific marker genes for categorization of beer-spoiling Lactobacillus brevis.

    Behr, Jürgen; Geissler, Andreas J; Preissler, Patrick; Ehrenreich, Armin; Angelov, Angel; Vogel, Rudi F


    The tolerance to hop compounds, which is mainly associated with inhibition of bacterial growth in beer, is a multi-factorial trait. Any approaches to predict the physiological differences between beer-spoiling and non-spoiling strains on the basis of a single marker gene are limited. We identified ecotype-specific genes related to the ability to grow in Pilsner beer via comparative genome sequencing. The genome sequences of four different strains of Lactobacillus brevis were compared, including newly established genomes of two highly hop tolerant beer isolates, one strain isolated from faeces and one published genome of a silage isolate. Gene fragments exclusively occurring in beer-spoiling strains as well as sequences only occurring in non-spoiling strains were identified. Comparative genomic arrays were established and hybridized with a set of L. brevis strains, which are characterized by their ability to spoil beer. As result, a set of 33 and 4 oligonucleotide probes could be established specifically detecting beer-spoilers and non-spoilers, respectively. The detection of more than one of these marker sequences according to a genetic barcode enables scoring of L. brevis for their beer-spoiling potential and can thus assist in risk evaluation in brewing industry. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Phylogeny of the glomeromycota (arbuscular mycorrhizal fungi): recent developments and new gene markers.

    Redecker, Dirk; Raab, Philipp


    The fungal symbionts of arbuscular mycorrhiza form a monophyletic group in the true Fungi, the phylum Glomeromycota. Fewer than 200 described species currently are included in this group. The only member of this clade known to form a different type of symbiosis is Geosiphon pyriformis, which associates with cyanobacteria. Because none of these fungi has been cultivated without their plant hosts or cyanobacterial partners, progress in obtaining multigene phylogenies has been slow and the nuclear-encoded ribosomal RNA genes have remained the only widely accessible molecular markers. rDNA phylogenies have revealed considerable polyphyly of some glomeromycotan genera that has been used to reassess taxonomic concepts. Environmental studies using phylogenetic methods for molecular identification have recovered an amazing diversity of unknown phylotypes, suggesting considerable cryptic species diversity. Protein gene sequences that have become available recently have challenged the rDNA-supported sister group relationship of the Glomeromycota with Asco/Basidiomycota. However the number of taxa analyzed with these new markers is still too small to provide a comprehensive picture of intraphylum relationships. We use nuclear-encoded rDNA and rpb1 protein gene sequences to reassess the phylogeny of the Glomeromycota and discuss possible implications.

  20. Gene flow among different taxonomic units: evidence from nuclear and cytoplasmic markers in Cedrus plantation forests.

    Fady, B; Lefèvre, F; Reynaud, M; Vendramin, G G; Bou Dagher-Kharrat, M; Anzidei, M; Pastorelli, R; Savouré, A; Bariteau, M


    Hybridization and introgression are important natural evolutionary processes that can be successfully investigated using molecular markers and open- and controlled-pollinated progeny. In this study, we collected open-pollinated seeds from Cedrus atlantica, Cedrus libani and C. libani x C. atlantica hybrids from three French-plantation forests. We also used pollen from C. libani and Cedrus brevifolia to pollinate C. atlantica trees. The progeny were analyzed using three different types of molecular markers: RAPDs, AFLPs and cpSSRs. Chloroplast DNA was found to be paternally inherited in Cedrus from the progeny of controlled-crosses. Heteroplasmy, although possible, could not be undoubtedly detected. There was no indication of strong reproductive isolating barriers among the three Mediterranean Cedrus taxa. Gene flow between C. atlantica and C. libani accounted for 67 to 81% of viable open-pollinated seedlings in two plantation forests. We propose that Mediterranean Cedrus taxa should be considered as units of a single collective species comprising two regional groups, North Africa and the Middle East. We recommend the use of cpSSRs for monitoring gene flow between taxa in plantation forests, especially in areas where garden specimens of one species are planted in the vicinity of selected seed-stands and gene-conservation reserves of another species.

  1. Genetic assessment of common bean (Phaseolus vulgaris L.) accessions by peroxidase gene-based markers.

    Nemli, Seda; Kaya, Hilal Betul; Tanyolac, Bahattin


    Peroxidase, a plant-specific oxidoreductase, is a heme-containing glycoprotein encoded by a large multigenic family in plants. Plant peroxidases (POXs, EC play important roles in many self-defense interactions in plants. Here, 67 common bean (Phaseolus vulgaris L.) genotypes were studied using a POX gene-based marker method. Comparison of POX genes could resolve evolutionary relationships in common bean. Eighty fragments were obtained with 20 primer pairs that amplified one (POX8c) to eight (ATP29) bands, with a mean of four bands per primer pair. The average (polymorphic information content) PIC value for the POX products was 0.40. The maximum variation (93%) was found between Turkey (#33) and India (#52) and between Antalya (#33) and India (#53). The minimum variation (0%) was found among four pairs: Bozdag (#2) and Karadeniz (#38), Kirklareli (#11) and Turkey (#15, 16, 43), Bandirma (#13) and Turkey (#15, 16, 43), and Kirklareli (#10) and Bandirma (#22). UPGMA was used to discriminate the common bean genotypes into five clusters, while STRUCTURE software was used to investigate the genetic population structure. The results showed that POX gene family markers can be used to study genotypic diversity and provide new information for breeding programs and common bean improvement practices. © 2013 Society of Chemical Industry.

  2. EST, COSII, and arbitrary gene markers give similar estimates of nucleotide diversity in cultivated tomato (Solanum lycopersicum L.).

    Labate, Joanne A; Robertson, Larry D; Wu, Feinan; Tanksley, Steven D; Baldo, Angela M


    Because cultivated tomato (Solanum lycopersicum L.) is low in genetic diversity, public, verified single nucleotide polymorphism (SNP) markers within the species are in demand. To promote marker development we resequenced approximately 23 kb in a diverse set of 31 tomato lines including TA496. Three classes of markers were sampled: (1) 26 expressed-sequence tag (EST), all of which were predicted to be polymorphic based on TA496, (2) 14 conserved ortholog set II (COSII) or unigene, and (3) ten published sequences, composed of nine fruit quality genes and one anonymous RFLP marker. The latter two types contained mostly noncoding DNA. In total, 154 SNPs and 34 indels were observed. The distributions of nucleotide diversity estimates among marker types were not significantly different from each other. Ascertainment bias of SNPs was evaluated for the EST markers. Despite the fact that the EST markers were developed using SNP prediction within a sample consisting of only one TA496 allele and one additional allele, the majority of polymorphisms in the 26 EST markers were represented among the other 30 tomato lines. Fifteen EST markers with published SNPs were more closely examined for bias. Mean SNP diversity observations were not significantly different between the original discovery sample of two lines (53 SNPs) and the 31 line diversity panel (56 SNPs). Furthermore, TA496 shared its haplotype with at least one other line at 11 of the 15 markers. These data demonstrate that public EST databases and noncoding regions are a valuable source of unbiased SNP markers in tomato.

  3. Endogenous gustatory responses and gene expression profile of stably proliferating human taste cells isolated from fungiform papillae.

    Hochheimer, Andreas; Krohn, Michael; Rudert, Kerstin; Riedel, Katja; Becker, Sven; Thirion, Christian; Zinke, Holger


    Investigating molecular mechanisms underlying human taste sensation requires functionally dedicated and at the same time proliferating human taste cells. Here, we isolated viable human fungiform taste papillae cells from biopsy samples, adenovirally transduced proliferation promoting genes, and obtained stably proliferating cell lines. Analysis of gene expression of 1 human taste cell line termed HTC-8 revealed that these cells express 13 TAS2R bitter taste receptor genes, CD36, OXTR encoding oxytocin receptor, as well as genes implicated with signal transduction and cell fate control. Bitter tastants triggered functionally distinct signaling pathways in HTC-8 cells. Salicin elicited phospholipase C-dependent calcium signaling and no cell depolarization. In contrast, stimulation with saccharin, aristolochic acid, or phenylthiocarbamide triggered cell depolarization and phospholipase C-independent calcium influx. Simultaneous stimulation with salicin and saccharin revealed that saccharin can enhance the phospholipase C-dependent response to salicin indicating crosstalk of signaling pathways. Our results show that HTC-8 cells are programmed to bitter taste reception but are also responsive to fatty acids, oxytocin, and somatosensory stimuli, whereas HTC-8 cells are insensitive to compounds representing other basic taste qualities.

  4. Development of a SCAR (sequence-characterised amplified region) marker for acid resistance-related gene in Lactobacillus plantarum.

    Liu, Shu-Wen; Li, Kai; Yang, Shi-Ling; Tian, Shu-Fen; He, Ling


    A sequence characterised amplified region marker was developed to determine an acid resistance-related gene in Lactobacillus plantarum. A random amplified polymorphic DNA marker named S116-680 was reported to be closely related to the acid resistance of the strains. The DNA band corresponding to this marker was cloned and sequenced with the induction of specific designed PCR primers. The results of PCR test helped to amplify a clear specific band of 680 bp in the tested acid-resistant strains. S116-680 marker would be useful to explore the acid-resistant mechanism of L. plantarum and to screen desirable malolactic fermentation strains.

  5. [The effects of stathmin on cell proliferation and tumor-related genes expressions in HCCLM3 cells].

    Gan, Lin; Li, Juan; Guo, Kun; Li, Yan; Shu, Hong; Wang, Li; Song, Jie; Liu, Yin-Kun


    To explore the biological function and possible underlying mechanism of stathmin gene during hepatocarcinogenesis. Three pairs of chemically synthesized small interfering RNA (siRNA) targeting on stathmin were transfected into HCCLM3 by LipofectamineTM 2000. After confirming the interfering effects of stathmin siRNAs through reverse transcription PCR and Western blotting, the HCCLM3 cells proliferation and apoptosis were detected by cell count kit-8 (CCK-8) and flow cytometry analysis, and the expressions of tumor-related genes (c-myc, c-fos, p53, etc) were observed by real-time PCR. Stathmin expression was effectively inhibited up to 90% by stathmin silencing in HCCLM3 cells (P is less than to 0.05) . By using CCK8 assay, it was shown that HCCLM3 cells proliferation were obviously depressed by 13.04%+/-0.10%, 28.10%+/-0.41% and 37.36%+/-2.15% at the time point of 24 h, 48 h and 72 h with the comparison to Mock group (F = 4.21, P is less than to 0.05). The results of flow cytometry demonstrated that the percentage of apoptotic cells was increased to 25.11%+/-1.62% in RNAi group, compared with 9.20 %+/-0.64 % in Mock group (F = 44.67, P is less than to 0.01). The results of real-time PCR showed that oncogenes c-myc and c-fos expressions were repressed, proliferation-associated gene ki-67 was down-regulated, and apoptosis-promoting gene caspase-3, bax and p53 were induced (P is less than to 0.05). Stathmin may promote cell proliferation, inhibit cell apoptosis and induce malignant transformation of hepatocytes by regulating some tumor-related genes expressions.

  6. Polymorphisms in the mitochondrial oxidative phosphorylation chain genes as prognostic markers for colorectal cancer

    Lascorz Jesus


    Full Text Available Abstract Background Currently, the TNM classification of malignant tumours based on clinicopathological staging remains the standard for colorectal cancer (CRC prognostication. Recently, we identified the mitochondrial oxidative phosphorylation chain as a consistently overrepresented category in the published gene expression profiling (GEP studies on CRC prognosis. Methods We evaluated associations of putative regulatory single nucleotide polymorphisms (SNPs in genes from the oxidative phosphorylation chain with survival and disease prognosis in 613 CRC patients from Northern Germany (PopGen cohort. Results Two SNPs in the 3′ untranslated region of UQCRB (complex III, rs7836698 and rs10504961, were associated with overall survival (HR = 0.52, 95% CI 0.32–0.85 and HR = 0.64, 95% CI 0.42–0.99, for TT carriers. These associations were restricted to the group of patients with cancer located in the colon (HR = 0.42, 95% CI 0.22–0.82 and HR = 0.46, 95% CI 0.25–0.83. Multivariate analysis indicated that both markers might act as independent prognostic markers. Additionally, the TT carriers were ~2 times more likely to develop tumours in the colon than in the rectum. Two SNPs in COX6B1 (complex IV were associated with lymph node metastasis in a dominant model (rs6510502, OR = 1.75, 95% CI 1.20–2.57; rs10420252, OR = 1.68, 95% CI 1.11–2.53; rs6510502 was associated also with distant metastasis (OR = 1.67, 95% CI 1.09–2.56 in a dominant model. Conclusions This is the first report suggesting that markers in genes from the mitochondrial oxidative chain might be prognostic factors for CRC. Additional studies replicating the presented findings are needed.

  7. MYC gene delivery to adult mouse utricles stimulates proliferation of postmitotic supporting cells in vitro.

    Joseph C Burns

    Full Text Available The inner ears of adult humans and other mammals possess a limited capacity for regenerating sensory hair cells, which can lead to permanent auditory and vestibular deficits. During development and regeneration, undifferentiated supporting cells within inner ear sensory epithelia can self-renew and give rise to new hair cells; however, these otic progenitors become depleted postnatally. Therefore, reprogramming differentiated supporting cells into otic progenitors is a potential strategy for restoring regenerative potential to the ear. Transient expression of the induced pluripotency transcription factors, Oct3/4, Klf4, Sox2, and c-Myc reprograms fibroblasts into neural progenitors under neural-promoting culture conditions, so as a first step, we explored whether ectopic expression of these factors can reverse supporting cell quiescence in whole organ cultures of adult mouse utricles. Co-infection of utricles with adenoviral vectors separately encoding Oct3/4, Klf4, Sox2, and the degradation-resistant T58A mutant of c-Myc (c-MycT58A triggered significant levels of supporting cell S-phase entry as assessed by continuous BrdU labeling. Of the four factors, c-MycT58A alone was both necessary and sufficient for the proliferative response. The number of BrdU-labeled cells plateaued between 5-7 days after infection, and then decreased ~60% by 3 weeks, as many cycling cells appeared to enter apoptosis. Switching to differentiation-promoting culture medium at 5 days after ectopic expression of c-MycT58A temporarily attenuated the loss of BrdU-labeled cells and accompanied a very modest but significant expansion of the sensory epithelium. A small number of the proliferating cells in these cultures labeled for the hair cell marker, myosin VIIA, suggesting they had begun differentiating towards a hair cell fate. The results indicate that ectopic expression of c-MycT58A in combination with methods for promoting cell survival and differentiation may restore

  8. MiR-27a Promotes Hepatocellular Carcinoma Cell Proliferation Through Suppression of its Target Gene Peroxisome Proliferator-activated Receptor γ

    Shuo Li; Jing Li; Bing-Yuan Fei; Dan Shao; Yue Pan; Zhan-Hao Mo; Bao-Zhen Sun


    Background:MicroRNAs (miRNAs) function as essential posttranscriptional modulators ofgene expression,and are involved in a wide range of physiologic and pathologic states,including cancer.Numerous miRNAs are deregulated in hepatocellular carcinoma (HCC).This study aimed to investigate the role of miR-27a in the development of HCC.Methods:The expression of MiR-27a was measured by quantitative real-time polymerase chain reaction (qRT-PCR).3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to examine changes in the viability of HepG2,Bel-7402,Bel-7404 hepatoma cell lines associated with up-regulation or down-regulation of miR-27a.A dual-luciferase activity assay was used to verify a target gene of miR-27a.Immunohistochemistry,qRT-PCR,Western blotting analysis,and cell cycle and apoptosis flow cytometric assays were used to elucidate the mechanism by which miR-27a modulates liver cancer cell proliferation.Results:The expression of miR-27a was significantly increased in HCC tissues and HepG2,Bel-7402,Bel-7404 hepatoma cell lines (P < 0.05).We also found that the down-regulation of miR-27a in HepG2 cells dramatically inhibited proliferation,blocked the G1 to S cell cycle transition and induced apoptosis (P < 0.05).In addition,miR-27a directly targeted the 3'-untranslated region of peroxisome proliferator-activated receptor γ (PPAR-γ),and ectopic miR-27a expression suppressed PPAR-γ expression on the mRNA and protein levels.The rosiglitazone-induced overexpression of PPAR-γ attenuated the effect of miR-27a in HCC cells.Conclusions:Our findings suggested that miRNA-27a promoted HCC cell proliferation by regulating PPAR-γ expression.MiR-27a may provide a potential therapeutic strategy for HCC treatment.

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

    Chanitra Thuwajit; Peti Thuwajit; Kazuhiko Uchida; Daoyot Daorueang; Sasithorn Kaewkes; Sopit Wongkham; Masanao Miwa


    AIM: To investigate the mechanism of fibroblast cell proliferation stimulated by the Opisthorchis viverrini excretory/secretory (ES) product.METHODS: NIH-3T3, mouse fibroblast cells were treated with O. viverrini ES product by non-contact co-cultured with the adult parasites. Total RNA from NIH-3T3 treated and untreated with O. viverrini was extracted, reverse transcribed and hybridized with the mouse 15K complementary DNA (cDNA) array. The result was analyzed by ArrayVision version 5 and GeneSpring version 5 softwares. After normalization, the ratios of gene expression of parasite treated to untreated NIH3T3 cells of 2-and more-fold upregulated was defined as the differentially expressed genes. The expression levels of the signal transduction genes were validated by semiquantitative SYBR-based real-time RT-PCR.RESULTS: Among a total of 15 000 genes/ESTs, 239genes with established cell proliferation-related function were 2 fold-and more-up-regulated by O. viverrini ES product compared to those in cells without exposure to the parasitic product. These genes were classified into groups including energy and metabolism, signal transduction, protein synthesis and translation, matrix and structural protein, transcription control, cell cycle and DNA replication. Moreover, the expressions of serinethreonine kinase receptor, receptor tyrosine kinase and collagen production-related genes were up-regulated by O. viverrini ES product. The expression level of signal transduction genes; pkC, pdgfrα, jak 1, eps 8, tgfβ 1/4,strap and h ras measured by real-time RT-PCR confirmed their expression levels to those obtained from cDNA array. However, only the up-regulated expression of pkC, eps 8 and tgfβ 1/4 which are the downstream signaling molecules of either epidermal growth factor (EGF) or transforming growth factor-β (TGF-β) showed statistical significance (P < 0.05). CONCLUSION: O. viverrini ES product stimulates the significant changes of gene expression in several

  10. [Gene pool differentiation between Altaic and trotting horse breeds inferred from ISSR-PCR marker data].

    Feofilov, A V; Bardukov, N V; Glazko, V I


    Using ISSR-PCR marker data, comparative analysis of the gene pools of Altaic and trotting horse breeds was carried out. Horse groups of different origin demonstrated differences in amplification spectra of DNA fragments flanked by inverted repeats of four microsatellites. Combinations of certain DNA fragments present in these profiles reproducibly distinguished genomes of the Altaic breed from the trotting breeds. Genetic differentiation between some trotting breeds, based on Nei genetic distance values, was found to be comparable to that between the groups of horses of Altaic breed from two different farms.

  11. Silencing of the Menkes copper-transporting ATPase (Atp7a) gene increases cyclin D1 protein expression and impairs proliferation of rat intestinal epithelial (IEC-6) cells.

    Gulec, Sukru; Collins, James F


    The Menkes copper-transporting ATPase (Atp7a) has dual roles in mammalian enterocytes: pumping copper into the trans-Golgi network (to support cuproenzyme synthesis) and across the basolateral membrane (to deliver dietary copper to the blood). Atp7a is strongly induced in the rodent duodenum during iron deprivation, suggesting that copper influences iron homeostasis. To investigate this possibility, Atp7a was silenced in rat intestinal epithelial (IEC-6) cells. Irrespective of its influence on iron homeostasis, an unexpected observation was made in the Atp7a knockdown (KD) cells: the cells grew slower (∼40% fewer cells at 96h) and were larger than negative-control shRNA-transfected cells. Lack of Atp7a activity thus perturbed cell cycle control. To elucidate a possible molecular mechanism, expression of two important cell cycle control proteins was assessed. Cyclin D1 (CD1) protein expression increased in Atp7a KD cells whereas proliferating-cell nuclear antigen (PCNA) expression was unaltered. Increased CD1 expression is consistent with impaired cell cycle progression. Expression of additional cell proliferation marker genes (p21 and Ki67) was also investigated; p21 expression increased, whereas Ki67 decreased, both consistent with diminished cell growth. Further experiments were designed to determine whether increased cellular copper content was the trigger for the altered growth phenotype of the Atp7a KD cells. Copper loading, however, did not influence the expression patterns of CD1, p21 or Ki67. Overall, these findings demonstrate that Atp7a is required for normal proliferation of IEC-6 cells. How Atp7a influences cell growth is unclear, but the underlying mechanism could relate to its roles in intracellular copper distribution or cuproenzyme synthesis. Copyright © 2014. Published by Elsevier GmbH.

  12. Transcription Profiles of Marker Genes Predict The Transdifferentiation Relationship between Eight Types of Liver Cell during Rat Liver Regeneration

    Xiaguang Chen


    Full Text Available Objective: To investigate the transdifferentiation relationship between eight types of liver cell during rat liver regeneration (LR. Materials and Methods: 114 healthy Sprague-Dawley (SD rats were used in this experimental study. Eight types of liver cell were isolated and purified with percoll density gradient centrifugation and immunomagentic bead methods. Marker genes for eight types of cell were obtained by retrieving the relevant references and databases. Expression changes of markers for each cell of the eight cell types were measured using microarray. The relationships between the expression profiles of marker genes and transdifferentiation among liver cells were analyzed using bioinformatics. Liver cell transdifferentiation was predicted by comparing expression profiles of marker genes in different liver cells. Results: During LR hepatocytes (HCs not only express hepatic oval cells (HOC markers (including PROM1, KRT14 and LY6E, but also express biliary epithelial cell (BEC markers (including KRT7 and KRT19; BECs express both HOC markers (including GABRP, PCNA and THY1 and HC markers such as CPS1, TAT, KRT8 and KRT18; both HC markers (KRT18, KRT8 and WT1 and BEC markers (KRT7 and KRT19 were detected in HOCs. Additionally, some HC markers were also significantly upregulated in hepatic stellate cells ( HSCs, sinusoidal endothelial cells (SECs , Kupffer cells (KCs and dendritic cells (DCs, mainly at 6-72 hours post partial hepatectomy (PH. Conclusion: Our findings indicate that there is a mutual transdifferentiation relationship between HC, BEC and HOC during LR, and a tendency for HSCs, SECs, KCs and DCs to transdifferentiate into HCs.

  13. Controversy Associated With the Common Component of Most Transgenic Plants – Kanamycin Resistance Marker Gene

    Srećko Jelenić


    Full Text Available Plant genetic engineering is a powerful tool for producing crops resistant to pests, diseases and abiotic stress or crops with improved nutritional value or better quality products. Currently over 70 genetically modified (GM crops have been approved for use in different countries. These cover a wide range of plant species with significant number of different modified traits. However, beside the technology used for their improvement, the common component of most GM crops is the neomycin phosphotransferase II gene (nptII, which confers resistance to the antibiotics kanamycin and neomycin. The nptII gene is present in GM crops as a marker gene to select transformed plant cells during the first steps of the transformation process. The use of antibiotic-resistance genes is subject to controversy and intense debate, because of the likelihood that clinical therapy could be compromised due to inactivation of the oral dose of the antibiotic from consumption of food derived from the transgenic plant, and because of the risk of gene transfer from plants to gut and soil microorganisms or to consumer’s cells. The present article discusses these possibilities in the light of current scientific knowledge.

  14. Codon-based phylogenetics introduces novel flagellar gene markers to oomycete systematics.

    Robideau, Gregg P; Rodrigue, Nicolas; André Lévesque, C


    Oomycete systematics has traditionally been reliant on ribosomal RNA and mitochondrial cytochrome oxidase sequences. Here we report the use of two single-copy protein-coding flagellar genes, PF16 and OCM1, in oomycete systematics, showing their utility in phylogenetic reconstruction and species identification. Applying a recently proposed mutation-selection model of codon substitution, the phylogenetic relationships inferred by flagellar genes are largely in agreement with the current views of oomycete evolution, whereas nucleotide- and amino acid-level models produce biologically implausible reconstructions. Interesting parallels exist between the phylogeny inferred from the flagellar genes and zoospore ontology, providing external support for the tree obtained using the codon model. The resolution achieved for species identification is ample using PF16, and quite robust using OCM1, and the described PCR primers are able to amplify both genes for a range of oomycete genera. Altogether, when analyzed with a rich codon substitution model, these flagellar genes provide useful markers for the oomycete molecular toolbox.

  15. Molecular characterization of RAPD and SCAR marker linked to the frog-eye leaf spot resistance gene in soybean


    Two fragments SCS3620 and SCS3580 of the co-dominant marker OPS03620&580 that were linked to the resistance gene of soybean frog-eye leaf spot have been completely sequenced.A significant insertion of 30 bp is the main reason of the polymorphism between the two fragments.The results of Southern hybridization indicate that SCS3620 derives from a single- or low-copy sequence and can be used as an RFLP probe.A co-dominant SCAR marker SCS3620&580 has been developed based on the sequences.The segregation of SCS3620&580 is similar to that of RAPD marker OPS03620&580.Significant polymorphism has been shown between resistant and susceptible genotypes when 62 soybean genotypes were surveyed for the SCAR marker.Therefore,the marker can be used in the resistance breeding of soybean frog-eye leaf spot by marker-assisted selection.

  16. MYC/MIZ1-dependent gene repression inversely coordinates the circadian clock with cell cycle and proliferation.

    Shostak, Anton; Ruppert, Bianca; Ha, Nati; Bruns, Philipp; Toprak, Umut H; Eils, Roland; Schlesner, Matthias; Diernfellner, Axel; Brunner, Michael


    The circadian clock and the cell cycle are major cellular systems that organize global physiology in temporal fashion. It seems conceivable that the potentially conflicting programs are coordinated. We show here that overexpression of MYC in U2OS cells attenuates the clock and conversely promotes cell proliferation while downregulation of MYC strengthens the clock and reduces proliferation. Inhibition of the circadian clock is crucially dependent on the formation of repressive complexes of MYC with MIZ1 and subsequent downregulation of the core clock genes BMAL1 (ARNTL), CLOCK and NPAS2. We show furthermore that BMAL1 expression levels correlate inversely with MYC levels in 102 human lymphomas. Our data suggest that MYC acts as a master coordinator that inversely modulates the impact of cell cycle and circadian clock on gene expression.

  17. Fine mapping of the rice bacterial blight resistance gene Xa-4 and its co-segregation marker


    An F2 population developed from the Xa-4 near isogenic lines,IR24 and IRBB4,was used for fine mapping of the rice bacterial blight resistance gene,Xa-4.Some restriction fragment length polymorphism (RFLP) markers on the high-density map constructed by Harushima et al.and the amplified DNA fragments homologous to the conserved domains of plant disease resistance (R) genes were used to construct the genetic linkage map around the gene Xa-4 by scoring susceptible individuals in the population.Xa-4 was mapped between the RFLP marker G181 and the polymerase chain reaction (PCR) marker M55.The R gene homologous fragment marker RS13 was found co-segregating with Xa-4 by analyzing all the plants in the population.This result opened an approach to map-based cloning of this gene,and marker RS13 can be applied to molecular marker-assisted selection of Xa-4 in rice breeding programs.

  18. Inheritance Analysis and Identification of SSR Markers Linked to Late Blight Resistant Gene in Tomato

    ZHU Hai-shan; WU Tao; ZHANG Zhen-xian


    Late blight caused by Phytophthora infestans is the most serious disease of tomato production in China. Studies on the genetics of resistance and identification of molecular markers are very useful for breeding late blight resistant varieties.The objective of this paper was to study the inheritance of late blight resistance and identify simple sequence repeat (SSR)markers associated with resistance allele in tomato (Lycopersicon esculentum Mill). The results came from an F2 progeny of 241 plants derived from a cross between 5# inbred line that is susceptible to late blight and a resistant accession CLN2037E. The late blight responses of F2 plants were tested by artificially inoculation of detached-leaflets in plate and natural infection assayed under greenhouse conditions. Both methods showed that the resistance is dominant and inherited as monogenic trait. Genetic mapping and linkage analysis showed that the late blight resistance gene Ph-ROL was located on chromosome 9 with a genetic distance of 5.7 cM to the SSR marker TOM236.

  19. Genetic transformation of Nannochloropsis oculata with a bacterial phleomycin resistance gene as dominant selective marker

    Ma, Xiaolei; Pan, Kehou; Zhang, Lin; Zhu, Baohua; Yang, Guanpin; Zhang, Xiangyang


    The gene ble from Streptoalloteichus hindustanus is widely used as a selective antibiotic marker. It can control the phleomycin resistance, and significantly increase the tolerance of hosts to zeocin. The unicellular marine microalga Nannochloropsis oculata is extremely sensitive to zeocin. We selected ble as the selective marker for the genetic transformation of N. oculata. After the algal cells at a density of 2×107 cells mL-1 was digested with 4% hemicellulase and 2% driselase for 1 h, the protoplasts accounted for 90% of the total. The ble was placed at the downstream of promoter HSP70A-RUBS2 isolated from Chlamydomonas reinhardtii, yielding a recombinant expression construct pMS188. The construct was transferred into the protoplasts through electroporation (1 kV, 15 μS). The transformed protoplasts were cultured in fresh f/2 liquid medium, and selected on solid f/2 medium supplemented with 500 ng mL-1 zeocin. The PCR result proved that ble existed in the transformants. Three transformants had been cultured for at least 5 generations without losing ble. Southern blotting analysis showed that the ble has been integrated into the genome of N. oculata. The ble will serve as a new dominant selective marker in genetic engineering N. oculata.

  20. Comparative Analysis of Cartilage Marker Gene Expression Patterns during Axolotl and Xenopus Limb Regeneration.

    Kazumasa Mitogawa

    Full Text Available Axolotls (Ambystoma mexicanum can completely regenerate lost limbs, whereas Xenopus laevis frogs cannot. During limb regeneration, a blastema is first formed at the amputation plane. It is thought that this regeneration blastema forms a limb by mechanisms similar to those of a developing embryonic limb bud. Furthermore, Xenopus laevis frogs can form a blastema after amputation; however, the blastema results in a terminal cone-shaped cartilaginous structure called a "spike." The causes of this patterning defect in Xenopus frog limb regeneration were explored. We hypothesized that differences in chondrogenesis may underlie the patterning defect. Thus, we focused on chondrogenesis. Chondrogenesis marker genes, type I and type II collagen, were compared in regenerative and nonregenerative environments. There were marked differences between axolotls and Xenopus in the expression pattern of these chondrogenesis-associated genes. The relative deficit in the chondrogenic capacity of Xenopus blastema cells may account for the absence of total limb regenerative capacity.

  1. Mapping of a BYDV resistance gene from Thinopyrum intermedium in wheat background by molecular markers

    张增艳; 辛志勇; 马有志; 陈孝; 徐琼芳; 林志珊


    The wheat line H960642 is a homozygous wheat-Thinopyrum intermedium translocation line with resistance to BYDV by genomie in situ hybridization (GISH) and RFLP analysis. The genomie DNA of Th. intermedium was used as a probe, and eonunon wheat genomie DNA as a blocking in GISH experiment. The results showed that the chromosome segments of Th. intermedium were transferred to the distal end of a pair of wheat chromosomes. RFLP analysis indicated that the transloeation line H960642 is a T7DS·7DL-7XL translocation by using 8 probes mapped on the homoeologous group 7 in wheat. The tranalocation breakpoint is located between Xpsr680 and Xpsr965 about 90—99 cM from the centromere. The RFLP markers psr680 and psr687 were closoly linked with the BYDV resistance gene. The gene is located on the distal end of 7XL around Xpsr680 and Xpsr687.

  2. Evaluation of Germplasm Using SSR Markers of Functional Genes in Rice

    ZHAO Yong; YANG Kai; Akbar Ali Cheema; WENG Yue-jin


    16 SSR (Simple sequence repeats) primers of functional genes in rice were used to detect genetic diversity among 23 accessions of rice germplasm from 5 countries in the world. The average number of alleles per SSR locus was 5.2 with a range from 2 to 10. Genetic similarities among the 23 rice accessions ranged from 0.13 to 0.64. UPGMA cluster analysis showed that the 23 rice accessions could be classified into two distinct classes at similarities with a coefficient of 0.13. The Japonicas from Brazil, Japan and China were classified into Class I, along with upland rice from Brazil. The Indicas from Pakistan and Korea were classified into Class Ⅱ. Consequently, the function of genes SSR markers could be used as a useful tool for measuring genetic diversity, assigning rice to geographical distribution, ecotype, and pedigree relationship.

  3. Comparative Analysis of Cartilage Marker Gene Expression Patterns during Axolotl and Xenopus Limb Regeneration.

    Mitogawa, Kazumasa; Makanae, Aki; Satoh, Ayano; Satoh, Akira


    Axolotls (Ambystoma mexicanum) can completely regenerate lost limbs, whereas Xenopus laevis frogs cannot. During limb regeneration, a blastema is first formed at the amputation plane. It is thought that this regeneration blastema forms a limb by mechanisms similar to those of a developing embryonic limb bud. Furthermore, Xenopus laevis frogs can form a blastema after amputation; however, the blastema results in a terminal cone-shaped cartilaginous structure called a "spike." The causes of this patterning defect in Xenopus frog limb regeneration were explored. We hypothesized that differences in chondrogenesis may underlie the patterning defect. Thus, we focused on chondrogenesis. Chondrogenesis marker genes, type I and type II collagen, were compared in regenerative and nonregenerative environments. There were marked differences between axolotls and Xenopus in the expression pattern of these chondrogenesis-associated genes. The relative deficit in the chondrogenic capacity of Xenopus blastema cells may account for the absence of total limb regenerative capacity.

  4. Altered expression of cell proliferation-related and interferon-stimulated genes in colon cancer cells resistant to SN38.

    Gongora, Céline; Candeil, Laurent; Vezzio, Nadia; Copois, Virginie; Denis, Vincent; Breil, Corinne; Molina, Franck; Fraslon, Caroline; Conseiller, Emmanuel; Pau, Bernard; Martineau, Pierre; Del Rio, Maguy


    Irinotecan is a topoisomerase I inhibitor widely used as an anticancer agent in the treatment of metastatic colon cancer. However, its efficacy is often limited by the development of resistance. We have isolated a colon carcinoma cell line, HCT116-SN6, which displays a 6-fold higher resistance to SN38, the active metabolite of irinotecan. In this paper, we studied the molecular mechanisms that cause resistance to SN38 in the HCT116-SN6 cell line. First, we analyzed proliferation, cell cycle distribution, apoptosis, topoisomerase I expression and activity in SN38-resistant (HCT116-SN6) and sensitive (HCT116-s cells). We showed that the SN38-induced apoptosis and the SN38-activated cell cycle checkpoints leading to G(2)/M cell cycle arrest were similar in both cell lines. Topoisomerase I expression and catalytic activity were also unchanged. Then, we compared mRNA expression profiles in the two cell lines using the Affymetrix Human Genome GeneChip arrays U133A and B. Microarray analysis showed that among the genes, which were differentially expressed in HCT116-s and HCT116-SN6 cells, 27% were related to cell proliferation suggesting that proliferation might be the main target in the development of resistance to SN38. This result correlates with the phenotypic observation of a reduced growth rate in HCT116-SN6 resistant cells. Furthermore, 29% of the overexpressed genes were Interferon Stimulated Genes and we demonstrate that their overexpression is, at least partially, due to endogenous activation of the p38 MAP kinase pathway in SN38 resistant cells. In conclusion, a slower cell proliferation rate may be a major cause of acquired resistance to SN38 via a reduction of cell cycle progression through the S phase which is mandatory for the cytotoxic action of SN38. This lower growth rate could be due to the endogenous activation of p38.

  5. Association of peroxisome proliferator-activated receptor single-nucleotide polymorphisms and gene-gene interactions with the lipoprotein(a)



    Objective To examine the associations of 10 singlenucleotide polymorphisms(SNPs)in peroxisome proliferator-activated receptor(PPARs)gene with lipoprotein(a)level,and to investigate if there is gene-gene interaction among the SNPs on lipoprotein(a)level.Methods Totally 644 subjects(234 men and 410 women)were enrolled from Prevention of Multiple Metabolic Disorders and Metabolic Syndrome Study Cohort,which was an urban community survey study conducted in Jiangsu province.Ten SNPs in PPARα(rs135539,rs4253778,

  6. Prognostic value of the apoptotic index analysed jointly with selected cell cycle regulators and proliferation markers in non-small cell lung cancer.

    Dworakowska, Dorota; Jassem, Ewa; Jassem, Jacek; Karmoliński, Andrzej; Lapiński, Mariusz; Tomaszewski, Dariusz; Rzyman, Witold; Jaśkiewicz, Kazimierz; Sworczak, Krzysztof; Grossman, Ashley B


    In a previous small series of surgically treated non-small cell lung cancer patients (NSCLC), we found that higher apoptotic index (AI) negatively influenced survival (Dworakowska D, Jassem E, Jassem J, Karmolinski A, Dworakowski R, Wirth T, et al. Clinical significance of apoptotic index in non-small cell lung cancer: correlation with p53, mdm2, pRb and p21WAF1/CIP1 protein expression. J Cancer Res Clin Oncol 2005; 131:617-623.). In this study we attempted to verify our previous finding in larger group of 170 NSCLC cases, additionally correlating AI to selected cell cycle regulators as well as a proliferation marker. Apoptosis was assessed with the use of the TUNEL technique, whereas the expression of p53, pRb, mdm2, p21(WAF1/CIP1), cyclin D1 and PCNA were assessed immunohistochemically. The mean and the median AI was 12 and 8, respectively. The expression of p53, pRb, mdm2, p21(WAF1/CIP1) proteins and cyclin D1 was found in 47%, 71%, 37%, 65% and 40% of cases, respectively. The mean and the median PCNA labeling index (PCNA LI) was 34 and 35, respectively. AI was not correlated with any patient characteristic or other tumor markers. In uni- and multivariate analysis AI, analysed separately or jointly with cell cycle regulators and PCNA LI, did not influence disease-free or over-all survival. However, patients with "very high AI/very high PCNA LI" had a particularly poor prognosis (P=0.001). Patients with "very low AI/negative pRb" phenotype survived for a shorter time in comparison to others (P=0.04). In addition, patients with the highest PCNA LI had a worse outcome in comparison to patients with the lowest PCNA LI (P=0.04), especially those with concomitant p53 protein expression (P=0.026) or lacking pRb protein expression (P=0.04). This study demonstrates that joint analysis of several factors involved in apoptosis, proliferation and cell cycle regulation, but not AI alone, might provide additional prognostic information in NSCLC patients.

  7. [Melanoma: surface markers as the first point of targeted delivery of therapeutic genes in multilevel gene therapy].

    Pleshkan, V V; Zinov'eva, M V; Sverdlov, E D


    Melanoma is one of the most malignant tumors, aggressively metastasizing by lymphatic and hematogenous routes. Due to the resistance of melanoma cells to many types of chemotherapy, this disease causes high mortality rate. High hopes are pinned on gene therapeutic approaches to melanoma treatment. At present, one of the main problems of the efficient use of the post-genomic generation therapeutic means is the lack of optimal techniques of delivery of foreign genetic material to the patient's target cells. Surface specific markers of melanoma cells can be considered as promising therapeutic targets. This review describes currently known melanoma specific receptors and its stem cells, as well as contains data on melanoma antigens presented on the cell surface by major histocompatibility complex proteins. The ability of surface proteins to internalize might be successfully used for the development of methods of targeted delivery of gene therapeutic constructs. In conclusion, a concept of multilevel gene therapy and the possible role therein of surface determinants as targets of gene systems delivery to the tumor are discussed.

  8. Reassessment of blood gene expression markers for the prognosis of relapsing-remitting multiple sclerosis.

    Michael Hecker

    Full Text Available Despite considerable advances in the treatment of multiple sclerosis, current drugs are only partially effective. Most patients show reduced disease activity with therapy, but still experience relapses, increasing disability, and new brain lesions. Since there are no reliable clinical or biological markers of disease progression, long-term prognosis is difficult to predict for individual patients. We identified 18 studies that suggested genes expressed in blood as predictive biomarkers. We validated the prognostic value of those genes with three different microarray data sets comprising 148 patients in total. Using these data, we tested whether the genes were significantly differentially expressed between patients with good and poor courses of the disease. Poor progression was defined by relapses and/or increase of disability during a two-year follow-up, independent of the administered therapy. Of 110 genes that have been proposed as predictive biomarkers, most could not be confirmed in our analysis. However, the G protein-coupled membrane receptor GPR3 was expressed at significantly lower levels in patients with poor disease progression in all data sets. GPR3 has therefore a high potential to be a biomarker for predicting future disease activity. In addition, we examined the IL17 cytokines and receptors in more detail and propose IL17RC as a new, promising, transcript-based biomarker candidate. Further studies are needed to better understand the roles of these receptors in multiple sclerosis and its treatment and to clarify the utility of GPR3 and IL17RC expression levels in the blood as markers of long-term prognosis.

  9. Inflammation markers predict zinc transporter gene expression in women with type 2 diabetes mellitus.

    Foster, Meika; Petocz, Peter; Samman, Samir


    The pathology of type 2 diabetes mellitus (DM) often is associated with underlying states of conditioned zinc deficiency and chronic inflammation. Zinc and omega-3 polyunsaturated fatty acids each exhibit anti-inflammatory effects and may be of therapeutic benefit in the disease. The present randomized, double-blind, placebo-controlled, 12-week trial was designed to investigate the effects of zinc (40 mg/day) and α-linolenic acid (ALA; 2 g/day flaxseed oil) supplementation on markers of inflammation [interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, C-reactive protein (CRP)] and zinc transporter and metallothionein gene expression in 48 postmenopausal women with type 2 DM. No significant effects of zinc or ALA supplementation were observed on inflammatory marker concentrations or fold change in zinc transporter and metallothionein gene expression. Significant increases in plasma zinc concentrations were observed over time in the groups supplemented with zinc alone or combined with ALA (P=.007 and P=.009, respectively). An impact of zinc treatment on zinc transporter gene expression was found; ZnT5 was positively correlated with Zip3 mRNA (Pzinc, while zinc supplementation abolished the relationship between ZnT5 and Zip10. IL-6 predicted the expression levels and CRP predicted the fold change of the ZnT5, ZnT7, Zip1, Zip7 and Zip10 mRNA cluster (Pzinc transporter and metallothionein gene expression support an interrelationship between zinc homeostasis and inflammation in type 2 DM.

  10. Clusters of conserved beta cell marker genes for assessment of beta cell phenotype.

    Geert A Martens

    Full Text Available BACKGROUND AND METHODOLOGY: The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse, rat and human tissues generated a panel of conserved beta cell biomarkers. This panel was then used to compare isolated versus laser capture microdissected beta cells, monitor adaptations of the beta cell phenotype to fasting, and retrieve possible conserved transcriptional regulators. PRINCIPAL FINDINGS: A panel of 332 conserved beta cell biomarker genes was found to discriminate both isolated and laser capture microdissected beta cells from all other examined cell types. Of all conserved beta cell-markers, 15% were strongly beta cell-selective and functionally associated to hormone processing, 15% were shared with neuronal cells and associated to regulated synaptic vesicle transport and 30% with immune plus gut mucosal tissues reflecting active protein synthesis. Fasting specifically down-regulated the latter cluster, but preserved the neuronal and strongly beta cell-selective traits, indicating preserved differentiated state. Analysis of consensus binding site enrichment indicated major roles of CREB/ATF and various nutrient- or redox-regulated transcription factors in maintenance of differentiated beta cell phenotype. CONCLUSIONS: Conserved beta cell marker genes contain major gene clusters defined by their beta cell selectivity or by their additional abundance in either neural cells or in immune plus gut mucosal cells. This panel can be used as a template to identify changes in the differentiated state of beta cells.

  11. Inactivation of the olfactory marker protein (OMP) gene in river dolphins and other odontocete cetaceans.

    Springer, Mark S; Gatesy, John


    Various toothed whales (Odontoceti) are unique among mammals in lacking olfactory bulbs as adults and are thought to be anosmic (lacking the olfactory sense). At the molecular level, toothed whales have high percentages of pseudogenic olfactory receptor genes, but species that have been investigated to date retain an intact copy of the olfactory marker protein gene (OMP), which is highly expressed in olfactory receptor neurons and may regulate the temporal resolution of olfactory responses. One hypothesis for the retention of intact OMP in diverse odontocete lineages is that this gene is pleiotropic with additional functions that are unrelated to olfaction. Recent expression studies provide some support for this hypothesis. Here, we report OMP sequences for representatives of all extant cetacean families and provide the first molecular evidence for inactivation of this gene in vertebrates. Specifically, OMP exhibits independent inactivating mutations in six different odontocete lineages: four river dolphin genera (Platanista, Lipotes, Pontoporia, Inia), sperm whale (Physeter), and harbor porpoise (Phocoena). These results suggest that the only essential role of OMP that is maintained by natural selection is in olfaction, although a non-olfactory role for OMP cannot be ruled out for lineages that retain an intact copy of this gene. Available genome sequences from cetaceans and close outgroups provide evidence of inactivating mutations in two additional genes (CNGA2, CNGA4), which imply further pseudogenization events in the olfactory cascade of odontocetes. Selection analyses demonstrate that evolutionary constraints on all three genes (OMP, CNGA2, CNGA4) have been greatly reduced in Odontoceti, but retain a signature of purifying selection on the stem Cetacea branch and in Mysticeti (baleen whales). This pattern is compatible with the 'echolocation-priority' hypothesis for the evolution of OMP, which posits that negative selection was maintained in the common

  12. Associations of VCAM-1 gene polymorphisms with obesity and inflammation markers.

    Yu, Gyeong Im; Jun, Sang Eun; Shin, Dong Hoon


    Among the inflammatory mediators involved in the pathogenesis of obesity, cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) stand out. The aim of this study was to investigate the associations of ICAM-1 and VCAM-1 gene variants with obesity and to investigate the associations between these genetic polymorphisms and CRP, UA, and WBC count. Four SNPs of the VCAM-1 gene (rs3176860, rs2392221, rs3917010 and rs3176879) and two SNPs of the ICAM-1 gene (rs281432 and rs5498) were analyzed in 181 control (18 obese (BMI ≥ 25) subjects. The SNPs were genotyped by direct sequencing. In allele frequency analysis, the G allelic frequency of rs3176860 in the VCAM-1 gene was lower in the obese group (30.9%) than in the controls (41.2%) (P = 0.007). The C allelic frequency of rs3917010 was lower in the obese group (18.1%) than in the control (25.1%) (P = 0.03). In the haplotype analysis of VCAM-1 gene, the ht1 (ACA) was higher and ht2 (GCC) was lower in the obese subjects than in the controls (P = 0.0057 and P = 0.037, respectively). In the obese group, participants carrying the G allele of rs3176860 of the VCAM-1 gene showed a higher percentage of segmented neutrophils and CRP levels than those carrying only the A allele (P = 0.028 and P = 0.042, respectively). The results of this study suggest that VCAM-1 gene variants may be related to obesity and inflammatory markers in the Korean population.

  13. Inheritance and identification of molecular markers associated with a novel dwarfing gene in barley

    Sun Genlou


    Full Text Available Abstract Background Dwarfing genes have widely been used in barley breeding program. More than 30 types of dwarfs or semidwarfs have been reported, but a few has been exploited in barley breeding because pleiotropic effects of dwarfing genes cause some undesired traits. The plant architecture of newly discovered dwarfing germplasm "Huaai 11" consisted of desirable agronomic traits such as shortened stature and early maturity. Genetic factor controlling the plant height in dwarf line Huaai 11 was investigated. Results The Huaai 11 was crossed with tall varieties Monker, Mpyt, Zhenongda 3, Zaoshu 3, Advance, Huadamai 1, Huadamai 6, Hyproly and Ris01508. All the F1 plants displayed tall trait. Both tall and dwarf plants appeared in all the F2 populations with a 3:1 segregation ratio, suggesting that dwarfism of Huaai 11 is controlled by a single recessive gene, btwd1. Allelism test indicated that this dwarfing gene in the Huaai 11 is nonallelic with the gene br, uzu, sdw1 and denso. Using a double haploid population derived from a cross of Huadamai 6 and Huaai 11 and SSR markers the novel dwarfing gene was mapped onto the long arm of chromosome 7H, and closely linked to Bmac031 and Bmac167 with genetic distance of 2.2 cM. Conclusion Huaai 11 is a new source of dwarf for broadening the genetic base of dwarfism. This dwarf source was controlled by a recessive dwarfing gene btwd1, was mapped onto the long arm of chromosome 7H.

  14. Genome-wide identification of R genes and exploitation of candidate RGA markers in rice

    WANG Xusheng; WU Weiren; JIN Gulei; ZHU Jun


    By scanning the whole genomic sequence of japonica rice using 45 known plant disease resistance (R) genes, we identified 2119 resistance gene homologs or analogs (RGAs) and verified that RGAs are not randomly distributed but tend to cluster in the rice genome. The RGAs were classified into 21 families according to their functional domain based on Hidden Markov model (HMM). By comparing the RGAs of japonica rice with the whole genomic sequence of indica rice, we found 702 RGAs allelic between the two subspecies and revealed that 671 (95.6%) of them have length difference (InDels) in their genomic sequences (including coding and non-coding regions) between the two subspecies, suggesting that RGAs are highly polymorphic between the two subspecies in rice. We also exploited 402 PCR-based and co-dominant candidate RGA markers by designing primer pairs on the regions flanking the InDels and validating them via e-PCR. The length differences of the candidate RGA markers between the two subspecies are from 1 to 742 bp, with an average of 10.26 bp. All related information of the RGAs is available from our web site (

  15. Prolactin promoter gene as marker assisted selection (MAS for the control of broodiness of Kampung chicken

    Tike Sartika


    Full Text Available Preliminary research about MAS (Marker Assisted Selection was conducted to detect broodiness trait of Kampung chicken. MAS currently is very important in situations, where the accuracy of selection is low, such as, traits with low heritability, e.g. broodiness trait and egg production. Prolactin promoter was selected as a marker gene for broodiness because it plays a critical part in the neuroendocrine cascade which is triggered at the onset of broodiness. DNA samples were collected from low and highbroodiness samples on basic population (G0 each 24 samples, and from selected population (G3 each 28 samples. As control population without broody behavior was used 16 samples White Leghorn (WL chicken. Prolactin promoter gene was amplified using polymerase chain reaction (PCR. PCR product was analyzed using electrophoresis agarose gel 2%. The results showed four types of bands represent in the Kampung chicken, three types called as wild type band and one type as the WL band. The chickens with low and high broodiness on G0 generation have 75 and 87.5% of wild type band while in the G3 generation was decreased to 25 and 75%. Conclusions of the research indicated that the selected breed of the Kampung chicken on G3 generation increased WL band like White Leghorn chicken as much as 31,25% from the G0 generation.

  16. Expression pattern and polymorphism of three microsatellite markers in the porcine CA3 gene

    Zheng Rong


    Full Text Available Abstract Carbonic anhydrase III (CA3 is an abundant muscle protein characteristic of adult type-1, slow-twitch, muscle fibres. In order to further understand the functions of the porcine CA3 protein in muscle, the temporal and spatial distributions of its gene product were analysed and the association between the presence of specific polymorphisms and carcass traits in the pig was also examined. Real-time PCR revealed that the CA3 mRNA expression showed no differences with age in skeletal muscles from Yorkshire pigs at postnatal day-1, month-2, and month-4. We provide the first evidence that CA3 is differentially expressed in the skeletal muscle of Yorkshire and Meishan pig breeds. In addition, the whole pig genomic DNA sequence of CA3 was investigated and shown to contain seven exons and six introns. Comparative sequencing of the gene from three pig breeds revealed the existence of microsatellite SJ160 in intron 5 and microsatellite SJ158 and a novel microsatellite marker that includes a tandem repeat of (TCn in intron 4. We also determined the allele number and frequencies of the three loci in seven pig breeds and found that they are low polymorphic microsatellite markers. Statistical analysis showed that the CA3 microsatellite polymorphism was associated with dressing percentage, internal fat rate, carcass length, rib number and backfat thickness in the pig.

  17. The dsdA gene from Escherichia coli provides a novel selectable marker for plant transformation.

    Erikson, Oskar; Hertzberg, Magnus; Näsholm, Torgny


    Plants are sensitive to D-serine, but functional expression of the dsdA gene, encoding D-serine ammonia lyase, from Escherichia coli can alleviate this toxicity. Plants, in contrast to many other organisms, lack the common pathway for oxidative deamination of D-amino acids. This difference in metabolism has major consequences for plant responses to D-amino acids, since several D-amino acids are toxic to plants even at relatively low concentrations. Therefore, introducing an enzyme specific for a phytotoxic D-amino acid should generate a selectable characteristic that can be screened. Here we present the use of the dsdA gene as a selectable marker for transformation of Arabidopsis. D-serine ammonia lyase catalyses the deamination of D-serine into pyruvate, water and ammonium. dsdA transgenic seedlings can be clearly distinguished from wild type, having an unambiguous phenotype immediately following germination when selected on D-serine containing medium. The dsdA marker allows flexibility in application of the selective agent: it can be applied in sterile plates, in foliar sprays or in liquid culture. Selection with D-serine resistance was compared with selection based on kanamycin resistance, and was found to generate similar transformation frequencies but also to be more unambiguous, more rapid and more versatile with respect to the way the selective agent can be supplied.

  18. The alteration of zinc transporter gene expression is associated with inflammatory markers in obese women.

    Noh, Hwayoung; Paik, Hee Young; Kim, Jihye; Chung, Jayong


    Obesity, a chronic inflammatory state, is associated with altered zinc metabolism. ZnT and Zip transporters are involved in the regulation of zinc metabolism. This study examined the relationships among obesity, zinc transporter gene expression, and inflammatory markers in young Korean women. The messenger RNA (mRNA) levels of leukocyte zinc transporters between obese (BMI = 28.3 ± 0.5 kg/m(2), n = 35) and nonobese (BMI = 20.7 ± 0.2 kg/m(2), n = 20) women aged 18-28 years were examined using quantitative real-time polymerase chain reaction. Inflammatory markers, such as C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-α), and interleukin (IL)-6, were measured in serum by enzyme immunoassay. ZnT1 and Zip1 were the most abundantly expressed zinc transporters in leukocytes. The mRNA levels of many zinc transporters (ZnT4, ZnT5, ZnT9, Zip1, Zip4, and Zip6) were significantly lower in obese women, and expression of these genes was inversely correlated with BMI and body fat percentage. In addition, inflammatory markers (CRP and TNF-α) were significantly higher in obese women. The mRNA levels of ZnT4, Zip1, and Zip6 were inversely correlated with CRP (P zinc transporters such as ZnT4, ZnT5, Zip1, and Zip6 (P zinc transporters may be altered in obese individuals. Changes in zinc transporters may also be related to the inflammatory state associated with obesity.

  19. Establishment of Relational Model of Congenital Heart Disease Markers and GO Functional Analysis of the Association between Its Serum Markers and Susceptibility Genes

    Min Liu


    Full Text Available Purpose. The purpose of present study was to construct the best screening model of congenital heart disease serum markers and to provide reference for further prevention and treatment of the disease. Methods. Documents from 2006 to 2014 were collected and meta-analysis was used for screening susceptibility genes and serum markers closely related to the diagnosis of congenital heart disease. Data of serum markers were extracted from 80 congenital heart disease patients and 80 healthy controls, respectively, and then logistic regression analysis and support vector machine were utilized to establish prediction models of serum markers and Gene Ontology (GO functional annotation. Results. Results showed that NKX2.5, GATA4, and FOG2 were susceptibility genes of congenital heart disease. CRP, BNP, and cTnI were risk factors of congenital heart disease (p<0.05; cTnI, hs-CRP, BNP, and Lp(a were significantly close to congenital heart disease (p<0.01. ROC curve indicated that the accuracy rate of Lp(a and cTnI, Lp(a and BNP, and BNP and cTnI joint prediction was 93.4%, 87.1%, and 97.2%, respectively. But the detection accuracy rate of the markers’ relational model established by support vector machine was only 85%. GO analysis suggested that NKX2.5, GATA4, and FOG2 were functionally related to Lp(a and BNP. Conclusions. The combined markers model of BNP and cTnI had the highest accuracy rate, providing a theoretical basis for the diagnosis of congenital heart disease.

  20. Production and processing studies on calpain-system gene markers for tenderness in Brahman cattle: 2. Objective meat quality.

    Cafe, L M; McIntyre, B L; Robinson, D L; Geesink, G H; Barendse, W; Pethick, D W; Thompson, J M; Greenwood, P L


    Effects and interactions of calpain-system tenderness gene markers on objective meat quality traits of Brahman (Bos indicus) cattle were quantified within 2 concurrent experiments at different locations. Cattle were selected for study from commercial and research herds at weaning based on their genotype for calpastatin (CAST) and calpain 3 (CAPN3) gene markers for beef tenderness. Gene marker status for mu-calpain (CAPN1-4751 and CAPN1-316) was also determined for inclusion in statistical analyses. Eighty-two heifer and 82 castrated male cattle with 0 or 2 favorable alleles for CAST and CAPN3 were studied in New South Wales (NSW), and 143 castrated male cattle with 0, 1, or 2 favorable alleles for CAST and CAPN3 were studied in Western Australia (WA). The cattle were backgrounded for 6 to 8 mo and grain-fed for 117 d (NSW) or 80 d (WA) before slaughter. One-half the cattle in each experiment were implanted with a hormonal growth promotant during feedlotting. One side of each carcass was suspended from the Achilles tendon (AT) and the other from the pelvis (tenderstretch). The M. longissimus lumborum from both sides and the M. semitendinosus from the AT side were collected; then samples of each were aged at 1 degrees C for 1 or 7 d. Favorable alleles for one or more markers reduced shear force, with little effect on other meat quality traits. The size of effects of individual markers varied with site, muscle, method of carcass suspension, and aging period. Individual marker effects were additive as evident in cattle with 4 favorable alleles for CAST and CAPN3 markers, which had shear force reductions of 12.2 N (P 0.05) of interactions between the gene markers, or between the hormonal growth promotant and gene markers for any meat quality traits. This study provides further evidence that selection based on the CAST or CAPN3 gene markers improves meat tenderness in Brahman cattle, with little if any detrimental effects on other meat quality traits. The CAPN1-4751 gene

  1. Sexually dimorphic gene expressions in eels: useful markers for early sex assessment in a conservation context

    Geffroy, Benjamin; Guilbaud, Florian; Amilhat, Elsa; Beaulaton, Laurent; Vignon, Matthias; Huchet, Emmanuel; Rives, Jacques; Bobe, Julien; Fostier, Alexis; Guiguen, Yann; Bardonnet, Agnès


    Environmental sex determination (ESD) has been detected in a range of vertebrate reptile and fish species. Eels are characterized by an ESD that occurs relatively late, since sex cannot be histologically determined before individuals reach 28 cm. Because several eel species are at risk of extinction, assessing sex at the earliest stage is a crucial management issue. Based on preliminary results of RNA sequencing, we targeted genes susceptible to be differentially expressed between ovaries and testis at different stages of development. Using qPCR, we detected testis-specific expressions of dmrt1, amh, gsdf and pre-miR202 and ovary-specific expressions were obtained for zar1, zp3 and foxn5. We showed that gene expressions in the gonad of intersexual eels were quite similar to those of males, supporting the idea that intersexual eels represent a transitional stage towards testicular differentiation. To assess whether these genes would be effective early molecular markers, we sampled juvenile eels in two locations with highly skewed sex ratios. The combined expression of six of these genes allowed the discrimination of groups according to their potential future sex and thus this appears to be a useful tool to estimate sex ratios of undifferentiated juvenile eels.

  2. Measuring gene flow from two birdsfoot trefoil (Lotus corniculatus) field trials using transgenes as tracer markers.

    De Marchis, F; Bellucci, M; Arcioni, S


    Genetic engineering is becoming a useful tool in the improvement of plants but concern has been expressed about the potential environmental risks of releasing genetically modified (GM) organisms into the environment. Attention has focused on pollen dispersal as a major issue in the risk assessment of transgenic crop plants. In this study, pollen-mediated dispersal of transgenes via cross-fertilization was examined. Plants of Lotus corniculatus L. transformed with either the Escherichia coli asparagine synthetase gene asnA or the beta-glucuronidase gene uidA, were used as the pollen donor. Nontransgenic plants belonging to the species L. corniculatus L., L. tenuis Waldst. and Kit. ex Willd, and L. pedunculatus Cav., were utilized as recipients. Two experimental fields were established in two areas of central Italy. Plants carrying the uidA gene were partially sterile, therefore only the asnA gene was used as a tracer marker. No transgene flow between L. corniculatus transformants and the nontransgenic L. tenuis and L. pedunculatus plants was detected. As regards nontransgenic L. corniculatus plants, in one location flow of asnA transgene was detected up to 18 m from the 1.8 m2 donor plot. In the other location, pollen dispersal occurred up to 120 m from the 14 m2 pollinating plot.

  3. [Relationship Between Molecular Marker of Western Main Pig H-FABP Gene and IMF Content.].

    Pang, Wei-Jun; Sun, Shi-Duo; Li, Ying; Chen, Guo-Dong; Yang, Gong-She


    By using 265 pigs from eight breeds including Duroc,Landrace,Large White,Neijiang,Rongchang,Hanjiang Black,Hanzhong White,Bamei and wild ones, the genetic variations of 5'-upstream region from and the second intron in porcine H-FABP gene were checked by PCR-RFLP molecular marker with HinfI, Hae III and MspI,and effect of H-FABP gene on IMF content was then analyzed by least square analysis.The results showed as follows:(1) 8 pig breeds and wild pig had polymorphism at Hinf I-RFLP site. In above detected breeds,large white,Bamei pig, Hanjiang Black,Hanzhong White pig breeds and wild pig presented low polymorphism while other breeds have mediate polymorphism;(2)Among the tested breeds only 4 Chinese local pig breeds had no polymorphism at the Hae III-RFLP and Msp I-RFLP sites,but Duroc,Landrace,Largewhite, Hanzhong White pig breeds and wild pig had polymorphism. Wild pig at the Hae III-RFLP , Landrace,Largewhite and wild pig at the Hae III-RFLP and Msp I-RFLP sites were low polymorphism,others were mediate polymorphism;(3) H-FABP gene increased IMF content significantly(p0.05). Genetic effect of H-FABP gene on IMF content were HH>Hh>hh,DD.

  4. Molecular Characterization and SNP Markers of the β-purothionin Gene in Einkorn Wheats

    LI Jing-qiong; ZHENG You-liang; WEI Yu-ming


    Forty-three gene sequences encoding purothionin were characterized from the three species or subspecies of einkorn wheats.These sequences contained 887 bp,among which 92 SNPs including 29 indel loci were detected,giving an average SNP frequency of one SNP per 9.64 bases.According to these sequences,5 SNP markers were successfully designed,which were used to mine the variations of purothionin genes of 102 einkorn wheat accessions.Based on the 5 detected SNP loci,102 einkorn wheat accessions could be divided into 21 haplotypes,among which 11 hapiotypes contained a single sample.Phylogenetic analysis indicated that the purothionin genes from einkorn wheats were more closely related to those from D genome than B genome.Seven out of the 43 gene sequences were assumed to be pseudogenes by the definition of containing in-frame stop codons and small insertions/deletions leading to frameshifi.In the remaining 36 amino acid sequences,the 8 Cys and Tyr-13 loci in the mature thionin domain which played important roles in the biological activities were all conserved,whereas there were some varieties occurred in some other important amino acid residues such as Lys and Arg.

  5. Efficient, Antibiotic Marker-Free Transformation of a Dicot and a Monocot Crop with Glutamate 1-Semialdehyde Aminotransferase Selectable Marker Genes.

    Ferradini, Nicoletta; Giancaspro, Angelica; Nicolia, Alessandro; Gadaleta, Agata; Veronesi, Fabio; Rosellini, Daniele


    Antibiotic-free, efficient in vitro selection in plant genetic engineering can improve risk perception and speed up pre-market scrutiny of genetically modified crops. We provide a protocol for genetic transformation of two important crops, durum wheat and alfalfa, using a bacterial and a plant-derived selectable marker gene encoding mutated, gabaculine-insensitive glutamate 1-semialdehyde aminotransferase (GSA) enzymes. These methods can potentially be applied, with minor adaptations, to many other monocot and dicot crop plants.

  6. Effect of type I collagen on the adhesion, proliferation, and osteoblastic gene expression of bone marrow-derived mesenchymal stem cells

    刘刚; 胡蕴玉; 赵建宁; 吴苏稼; 熊卓; 吕荣


    Objective: To investigate the effects of porous poly lactide-co-glycolide (PLGA) modified by type I collagen on the adhesion, proliferation, and differentiation of rabbit marrow-derived mesenchymal stem cells (MSCs). Methods: The third generation MSCs isolated from mature rabbits by density gradient centrifugation were cultured at different initial concentrations on 0.3 cm×1.2 cm×2.0 cm 3-D porous PLGA coated by type I collagen in RPMI 1640 containing 10% fetal calf serum, while cultured on PLGA without type I collagen as control. The cells adhesive and proliferative behavior at 7, 14, and 21 days after inoculation was assessed by determining the incorporation rate of [3H]-TdR. In order to examine MSCs differentiation, the expression of osteoblasts marker genes, osteocalcin (OCN), alkaline phosphatase (ALP), osteopontin (OPN) mRNA, were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), and further more, the cell morphology at 21 days was also observed by scanning electron microscope (SEM). Results: Type I collagen promoted cell adhesion on PLGA. The valve was significantly higher than controls (6 h, 2144 cpm±141cpm vs. 1797 cpm±118 cpm, P=0.017; 8 h, 2311 cpm±113 cpm vs. 1891 cpm±103 cpm, P=0.01). The cells which cultured on PLGA coated with type I collagen showed significantly higher cell proliferation than controls on the 7th day (1021 cpm±159 cpm vs. 451 cpm±67 cpm, P=0.002), the 14th day (1472 cpm±82 cpm vs. 583 cpm±67 cpm, P<0.001) and 21th day (1728 cpm±78 cpm vs. 632 cpm±55 cpm, P<0.001). Osteoblasts markers, OCN, ALP, OPN mRNA, were all detected on PLGA coated by type I collagen on the 21th day, but OCN, OPN mRNA could not be found in controls. Spindle and polygonal cells well distributed on the polymer coated by type I collagen while cylindric or round cells in controls. Conclusions: Type I collagen is effective in promoting the adhesion, proliferation and differentiation of MSCs on PLGA.

  7. Inhibition of cell proliferation and in vitro markers of angiogenesis by indole-3-carbinol, a major indole metabolite present in cruciferous vegetables.

    Wu, Hsiao-Ting; Lin, Shyh-Hsiang; Chen, Yue-Hwa


    A variety of studies have suggested a cancer protective role of cruciferous vegetables. In the present study, we investigated the effect of indole-3-carbinol (I3C), a major indole metabolite in cruciferous vegetables, on cell proliferation and in vitro markers of angiogenesis in phorbol myristate acetate (PMA)-stimulated endothelial EA hy926 cells. The results showed that I3C inhibited the growth of EA hy926 cells in a concentration-dependent manner. The capillary-like tube formation by PMA-activated endothelial cells was significantly suppressed by I3C, and such inhibition was associated with decreased vascular endothelial growth factor (VEGF) and increased interleukin-8 (IL-8) secretion, but not with the expression of VEGF receptor-2 protein. Additionally, gelatin zymography analysis indicated that I3C suppressed activities of matrix metalloproteinases-2 (MMP-2) and MMP-9 stimulated by PMA. These results suggest that the dietary I3C may be useful in the treatment of human cancers and angiogenic diseases.

  8. Evaluation of cell proliferation, apoptosis, and dna-repair genes as potential biomarkers for ethanol-induced cns alterations

    Hicks Steven D


    Full Text Available Abstract Background Alcohol use disorders (AUDs lead to alterations in central nervous system (CNS architecture along with impaired learning and memory. Previous work from our group and that of others suggests that one mechanism underlying these changes is alteration of cell proliferation, apoptosis, and DNA-repair in neural stem cells (NSCs produced as a consequence of ethanol-induced effects on the expression of genes related to p53-signaling. This study tests the hypothesis that changes in the expression of p53-signaling genes represent biomarkers of ethanol abuse which can be identified in the peripheral blood of rat drinking models and human AUD subjects and posits that specific changes may be correlated with differences in neuropsychological measures and CNS structure. Results Remarkably, microarray analysis of 350 genes related to p53-signaling in peripheral blood leukocytes (PBLs of binge-drinking rats revealed 190 genes that were significantly altered after correcting for multiple testing. Moreover, 40 of these genes overlapped with those that we had previously observed to be changed in ethanol-exposed mouse NSCs. Expression changes in nine of these genes were tested for independent confirmation by a custom QuantiGene Plex (QGP assay for a subset of p53-signaling genes, where a consistent trend for decreased expression of mitosis-related genes was observed. One mitosis-related gene (Pttg1 was also changed in human lymphoblasts cultured with ethanol. In PBLs of human AUD subjects seven p53-signaling genes were changed compared with non-drinking controls. Correlation and principal components analysis were then used to identify significant relationships between the expression of these seven genes and a set of medical, demographic, neuropsychological and neuroimaging measures that distinguished AUD and control subjects. Two genes (Ercc1 and Mcm5 showed a highly significant correlation with AUD-induced decreases in the volume of the left

  9. Evaluation of cell proliferation, apoptosis, and DNA-repair genes as potential biomarkers for ethanol-induced CNS alterations.

    Hicks, Steven D; Lewis, Lambert; Ritchie, Julie; Burke, Patrick; Abdul-Malak, Ynesse; Adackapara, Nyssa; Canfield, Kelly; Shwarts, Erik; Gentile, Karen; Meszaros, Zsuzsa Szombathyne; Middleton, Frank A


    Alcohol use disorders (AUDs) lead to alterations in central nervous system (CNS) architecture along with impaired learning and memory. Previous work from our group and that of others suggests that one mechanism underlying these changes is alteration of cell proliferation, apoptosis, and DNA-repair in neural stem cells (NSCs) produced as a consequence of ethanol-induced effects on the expression of genes related to p53-signaling. This study tests the hypothesis that changes in the expression of p53-signaling genes represent biomarkers of ethanol abuse which can be identified in the peripheral blood of rat drinking models and human AUD subjects and posits that specific changes may be correlated with differences in neuropsychological measures and CNS structure. Remarkably, microarray analysis of 350 genes related to p53-signaling in peripheral blood leukocytes (PBLs) of binge-drinking rats revealed 190 genes that were significantly altered after correcting for multiple testing. Moreover, 40 of these genes overlapped with those that we had previously observed to be changed in ethanol-exposed mouse NSCs. Expression changes in nine of these genes were tested for independent confirmation by a custom QuantiGene Plex (QGP) assay for a subset of p53-signaling genes, where a consistent trend for decreased expression of mitosis-related genes was observed. One mitosis-related gene (Pttg1) was also changed in human lymphoblasts cultured with ethanol. In PBLs of human AUD subjects seven p53-signaling genes were changed compared with non-drinking controls. Correlation and principal components analysis were then used to identify significant relationships between the expression of these seven genes and a set of medical, demographic, neuropsychological and neuroimaging measures that distinguished AUD and control subjects. Two genes (Ercc1 and Mcm5) showed a highly significant correlation with AUD-induced decreases in the volume of the left parietal supramarginal gyrus and

  10. Angiotensinogen gene silencing reduces markers of inflammation and lipid accumulation in adipocytes

    Wenting eXin


    Full Text Available Inflammatory adipokines secreted from adipose tissue are major contributors to obesity-associated inflammation and other metabolic dysfunctions. We and others have recently documented the contribution of adipose tissue renin-angiotensin system (RAS to the pathogenesis of obesity, inflammation and insulin resistance. We hypothesized that adipocyte-derived angiotensinogen (Agt plays a critical role in adipogenesis and/or lipogenesis as well as inflammation. This was tested using 3T3-L1 adipocytes, stably transfected with Agt-shRNA or scrambled Sc-shRNAcas a control. Transfected preadipocytes were differentiated and used to investigate the role of adipose Agt through microarray and PCR analyses and adipokine profiling. As expected, Agt gene silencing significantly reduced the expression of Agt and its hormone product angiotensin II (Ang II, as well as lipid accumulation in 3T3-L1 adipocytes. Microarray studies identified several genes involved in lipid metabolism and inflammatory pathways which were down-regulated by Agt gene inactivation, such as glycerol-3-phosphate dehydrogenase 1 (Gpd1, serum amyloid A 3 (Saa3, nucleotide-binding oligomerization domain containing 1 (Nod1 and signal transducer and activator of transcription 1 (Stat1. Mouse adipogenesis PCR arrays revealed lower expression levels of adipogenic/lipogenic genes such as peroxisome proliferator activated receptor gamma (Pparg, sterol regulatory element binding transcription factor 1 (Srebf1, adipogenin (Adig, and fatty acid binding protein 4 (Fabp4. Further, silencing of Agt gene significantly lowered expression of pro-inflammatory adipokines including interleukin-6 (IL-6, tumor necrosis factor-alpha (TNF-α, and monocyte chemotactic protein-1 (MCP-1. In conclusion, this study directly demonstrates critical effects of Agt in adipocyte metabolism and inflammation and further support a potential role for adipose Agt in the pathogenesis of obesity-associated metabolic alterations.

  11. Analysis of angiogenic markers in oral squamous cell carcinoma-gene and protein expression.

    Jung, Susanne; Sielker, Sonja; Purcz, Nikolai; Sproll, Christoph; Acil, Yahya; Kleinheinz, Johannes


    Therapeutic strategies attacking oral squamous cell carcinoma have not essentially succeeded to improve long-term prognosis and overall survival over the last decades. Therefore, in this study, we aimed to illuminate the molecular regulation of angiogenesis in this tumour entity in order to demask novel markers of prognosis or therapeutic approach. A panel of significant transcriptional alterations in angiogenic genes of 83 cancer samples was established by comparison to 30 samples of healthy oral mucosa with microarray technique. Immunohistochemistry (IHC) was performed to trace the signalling cascade from gene to protein level. A distinctive expression profile of VEGFA, EFNB2, PECAM1/CD31, ANGPT1 and ANGPT2 was revealed: VEGFA, EFNB2, and ANGPT2 were found overexpressed in 84 % to 95 % of tumour samples. In contrast, the expression of CD31 and ANGPT1 was downregulated in 80 % to 95 % of tumour samples. IHC confirmed results of the microarray analysis. Tumours with lymphatic spread showed higher gene expression rates of VEGFA, EFNB2 and ANGPT2 in moderately differentiated tumours and of VEGFA and EFNB2 in small tumours, respectively. The ANGPT1/ ANGPT2 transcription ratio was found decreased in larger tumours and especially in tumours without lymphatic spread. A characteristic expression profile of angiogenic markers was established. The specific overexpression of EFNB2 in small tumours with lymphatic spread and the typical decrease of the ANGPT1/ ANGPT2 ratio in larger tumours give weight to EFNB2 and angiopoietins as prognostic factors and potential therapeutic targets.

  12. [Knock-down of apollon gene by antisense oligodeoxynucleotide inhibits the proliferation of Lovo cells and enhances chemo-sensitivity].

    He, Jin-hua; Zhang, Xiao-ying; Wu, Feng-yun; Liao, Xiao-li; Wang, Wei; Jiang, Jian-wei


    In this study, the effects of apollon antisense oligodeoxynucleotide (ASODN) on the proliferation and apoptosis of human Lovo cells in vitro were investigated. Apollon ASODN was incubated with human colorectal Lovo cells for 48 h, the proliferation inhibition and the clone forming rates were detected by WST method and clone formation assay, respectively. The expression of apollon mRNA was analyzed by real time fluorescent quantitative reverse transcription polymerase chain reaction. The percentage of apoptotic cells and cell cycle distribution were determined by flow cytometry. The morphology of apoptotic cells was examined by fluorescence microscope. Lovo cells incubated with apollon ASODN combined with 5-fluorouracil (5-FU), cisplatin (DDP) or epirubicin (EPI) of different concentrations, cell proliferation inhibition rates were detected with WST method and IC50 was calculated. It was found that ASODN targeting apollon gene could all suppress the growth of Lovo cells and induce apoptosis of these cells significantly (P 5-FU, DDP and EPI on Lovo cells combined with apollon ASODN (0.08 micromol x L(-1)) were enhanced independently compared with single 5-FU, DDP and EPI groups, and the sensitivity enhanced about 2.58, 4.47, and 5.33 times respectively. It can be concluded that ASODN targeting apollon can suppress the expression of apollon mRNA, and inhibit the proliferation, induce apoptosis, arrest cell cycle at S phase of colorectal cancer Lovo cells in vitro and enhance the chemo-sensitivity to 5-FU, DDP and EPI.

  13. Eukaryotic Expression of Human Arresten Gene and Its Effect on the Proliferation of Vascular Smooth Muscle Cells

    SHANG Dan; ZHENG Qichang; SONG Zifang; LI Yiqing; WANG Xiedan; GUO Xingjun


    The eukaryotic expression of human arresten geneand its effect on the proliferation of in vitro cultured vascular smooth cells (VSMCs) in vitro were investigated. COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome. Forty-eight h after transfection, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of arresten mRNA in the cells,while Western blot assay was applied to detect the expression of arresten protein in concentrated supernatant. Primary VSMCs from thoracic aorta of male Sprague-Dawley rats were cultured using the tissue explant method, and identified by immunohistochemical staining with a smooth muscle-specific anti-αactin monoclonal antibody before serial subcultivation. VSMCs were then co-cultured with the concentrated supernatant and their proliferation was detected using Cell Counting Kit-8 (CCK-8) in vitro. The results showed that RT-PCR revealed that the genome of arresten-transfected cells contained a 449 bp specific fragment of arresten gene, suggesting the successful transfection. Successful protein expression in supernatants was confirmed by Western blot. CCK-8 assay showed that the proliferation of VSMCs were inhibited significantly by arresten protein as compared with control cells (F=40.154, P<0.01). It was concluded that arresten protein expressed in eukaryotic cells can inhibit proliferation of VSMCs effectively in vitro, which would provide possibility to the animal experiments.

  14. (SSR) markers



    Jul 30, 2014 ... Simple sequence repeats (SSRs) are the most widely used marker system for plant variety characterization and ... gene tagging in marker assisted breeding and gene cloning in .... PLS-2 and PAU Selection Long) to 1.00 (between PC. 2062 and .... Comparative analyses of genetic diversities within tomato.

  15. Inhibition of primordial germ cell proliferation by the medaka male determining gene Dmrt1bY

    Hornung Ute


    Full Text Available Abstract Background Dmrt1 is a highly conserved gene involved in the determination and early differentiation phase of the primordial gonad in vertebrates. In the fish medaka dmrt1bY, a functional duplicate of the autosomal dmrt1a gene on the Y-chromosome, has been shown to be the master regulator of male gonadal development, comparable to Sry in mammals. In males mRNA and protein expression was observed before morphological sex differentiation in the somatic cells surrounding primordial germ cells (PGCs of the gonadal anlage and later on exclusively in Sertoli cells. This suggested a role for dmrt1bY during male gonad and germ cell development. Results We provide functional evidence that expression of dmrt1bY leads to negative regulation of PGC proliferation. Flow cytometric measurements revealed a G2 arrest of dmrt1bY expressing cells. Interestingly, also non-transfected cells displayed a significantly lower fraction of proliferating cells, pointing to a possible non-cell autonomous action of dmrt1bY. Injection of antisense morpholinos led to an increase of PGCs in genetically male embryos due to loss of proliferation inhibition. Conclusion In medaka, dmrt1bY mediates a mitotic arrest of PGCs in males prior to testes differentiation at the sex determination stage. This occurs possibly via a cross-talk of Sertoli cells and PGCs.

  16. Development of molecular markers tightly linked to Pvr4 gene in pepper using next-generation sequencing.

    Devran, Zübeyir; Kahveci, Erdem; Özkaynak, Ercan; Studholme, David J; Tör, Mahmut

    It is imperative to identify highly polymorphic and tightly linked markers of a known trait for molecular marker-assisted selection. Potyvirus resistance 4 (Pvr4) locus in pepper confers resistance to three pathotypes of potato virus Y and to pepper mottle virus. We describe the use of next-generation sequencing technology to generate molecular markers tightly linked to Pvr4. Initially, comparative genomics was carried out, and a syntenic region of tomato on chromosome ten was used to generate PCR-based markers and map Pvr4. Subsequently, the genomic sequence of pepper was used, and more than 5000 single-nucleotide variants (SNVs) were identified within the interval. In addition, we identified nucleotide binding site-leucine-rich repeat-type disease resistance genes within the interval. Several of these SNVs were converted to molecular markers desirable for large-scale molecular breeding programmes.

  17. Pro12Ala polymorphism in the peroxisome proliferator-activated receptor-gamma (PPARgamma) gene in inflammatory bowel disease.

    Atug, Ozlen; Tahan, Veysel; Eren, Fatih; Tiftikci, Arzu; Imeryuz, Nese; Hamzaoglu, Hulya Over; Tozun, Nurdan


    Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has recently been implicated as an endogenous regulator of cellular proliferation and inflammation. Impaired expression of PPAR-gamma in colonic epithelial cells in ulcerative colitis (UC) and increased expression in hypertrophic mesenteric adipose tissue in Crohn's disease (CD) have been reported. Furthermore, PPAR-gamma ligands have been shown to inhibit tissue injury associated with immune activation in UC. Any mutation in PPAR-gamma gene may be responsible for the increase in inflammatory mediators and hence the perpetuation of inflammation in inflammatory bowel disease (IBD) patients. One common polymorphism in PPAR-gamma gene is proline to alanine substitution (Pro12Ala) which results from a CCA to GCA missense substitution in codon 12 of exon 2 of the PPAR-gamma gene. In this study, we aimed to explore Pro12Ala polymorphism in PPAR-gamma gene in IBD in Turkish patients. 69 patients with CD, 45 with UC and 100 controls of similar age and sex were studied. Genomic DNA was isolated from peripheral blood leucocytes and mutagenically separated-polymerase chain reaction (PCR) analyses were performed to determine the Pro12Ala polymorphism of the PPAR-gamma gene. We observed no significant differences in the frequency of the Pro12Ala polymorphism in the PPAR-gamma gene among subjects with CD, UC and controls (15.9%, 15.5% and 13%, respectively, p>0.05). These results suggest that Pro12Ala polymorphism in the PPAR-gamma gene relates neither to the risk of the development of inflammatory bowel disease nor to the clinical subtypes of CD in the Turkish population.

  18. Genome-Wide Gene Expression Profile Analyses Identify CTTN as a Potential Prognostic Marker in Esophageal Cancer


    Aim Esophageal squamous cell carcinoma (ESCC) is one of the most common fatal malignances of the digestive tract. Its prognosis is poor mainly due to the lack of reliable markers for early detection and prognostic prediction. Here we aim to identify the molecules involved in ESCC carcinogenesis and those as potential markers for prognosis and as new molecular therapeutic targets. Methods We performed genome-wide gene expression profile analyses of 10 primary ESCCs and their adjacent normal ti...

  19. Identification of Modulators of the Nuclear Receptor Peroxisome Proliferator-Activated Receptor α (PPARα) in a Mouse Liver Gene Expression Compendium

    The nuclear receptor family member peroxisome proliferator-activated receptor α (PPARα) is activated by therapeutic hypolipidemic drugs and environmentally-relevant chemicals to regulate genes involved in lipid transport and catabolism. Chronic activation of PPARα in rodents inc...

  20. Glial cell derived neurotrophic factor induces spermatogonial stem cell marker genes in chicken mesenchymal stem cells.

    Boozarpour, Sohrab; Matin, Maryam M; Momeni-Moghaddam, Madjid; Dehghani, Hesam; Mahdavi-Shahri, Naser; Sisakhtnezhad, Sajjad; Heirani-Tabasi, Asieh; Irfan-Maqsood, Muhammad; Bahrami, Ahmad Reza


    Mesenchymal stem cells (MSCs) are known with the potential of multi-lineage differentiation. Advances in differentiation technology have also resulted in the conversion of MSCs to other kinds of stem cells. MSCs are considered as a suitable source of cells for biotechnology purposes because they are abundant, easily accessible and well characterized cells. Nowadays small molecules are introduced as novel and efficient factors to differentiate stem cells. In this work, we examined the potential of glial cell derived neurotrophic factor (GDNF) for differentiating chicken MSCs toward spermatogonial stem cells. MSCs were isolated and characterized from chicken and cultured under treatment with all-trans retinoic acid (RA) or glial cell derived neurotrophic factor. Expression analysis of specific genes after 7days of RA treatment, as examined by RT-PCR, proved positive for some germ cell markers such as CVH, STRA8, PLZF and some genes involved in spermatogonial stem cell maintenance like BCL6b and c-KIT. On the other hand, GDNF could additionally induce expression of POU5F1, and NANOG as well as other genes which were induced after RA treatment. These data illustrated that GDNF is relatively more effective in diverting chicken MSCs towards Spermatogonial stem cell -like cells in chickens and suggests GDNF as a new agent to obtain transgenic poultry, nevertheless, exploitability of these cells should be verified by more experiments.

  1. A DNA marker for the Bt-10 common bunt resistance gene in wheat.

    Demeke, T; Laroche, A; Gaudet, D A


    The Bt-10 bunt gene confers resistance to most races of the common bunt fungi, Tilletia tritici and T. laevis. The RAPD technique, employing a total of 965 decamer primers, was used to identify polymorphic markers between resistant (BW553) and susceptible ('Neepawa") near-isogenic lines. Primer 196 (5' CTC CTC CCC C 3') produced a 590 base pair (bp) reproducible fragment only in the resistant near-isogenic line. The 590-bp DNA fragment was present in all the 22 wheat cultivars known to carry the Bt-10 resistance gene and also in 15 resistant F2 lines obtained from a cross between the resistant parent, BW553, and the susceptible parent, 'Neepawa'. The 590-bp fragment was absent in 16 susceptible cultivars tested and in 15 susceptible F2 lines obtained from the cross described above. These results suggest a close linkage between the presence of the 590-bp fragment and the Bt-10 resistance gene. Primer 372 (5' CCC ACT GAC G 3') amplified a 1.0-kilobase (kb) fragment that was present only in the susceptible near-isogenic line. This 1.0-kb fragment was present in 13 of the 16 susceptible cultivars and in 13 of the 15 susceptible F2 lines. However, the primer also amplified the 1.0-kb fragment in some resistant cultivars and resistant F2 lines, suggesting a looser linkage between the occurrence of the fragment and the susceptible allele.

  2. Development of new gene-specific markers associated with salt tolerance for mungbean (Vigna radiata L. Wilczek

    Nirmala Sehrawat


    Full Text Available Thirty eight novel microsatellite markers (SSRs specific to candidate genes involved in salt tolerance were developed for detection of genetic variations in 12 mungbean genotypes variably adapted to salt stress. A 100 out of 124 putative alleles were found polymorphic between wild and cultivated genotypes (inter-specific, 80.65%, 65 were within cultivars of mungbean (intra-specific, 52.42% and 52 were within wild genotypes (inter-specific, 41.94%. The polymorphism varied from 86.84% to 100%, while the number of polymorphic alleles ranged from 1 to 4 with an average value of 2.63 per locus. The polymorphism information content (PIC values ranged from 0.326 to 0.875 with an average value of 0.671, which shows their effectiveness in genetic analysis. Cluster analysis resulted in the distribution of salt tolerant and susceptible genotypes in separate groups which revealed the presence of inherent variations among mungbean cultivars. These variations were explored effectively for SSR markers studies. The developed SSR markers may help along with already available markers to execute further research on mungbean. The markers may be coupled with specific loci linked with salt tolerance. The developed markers will help to identify the QTLs (quantitative trait loci or other important genes. These markers can also be utilized for testing the purity of hybrids or diversity assessment of Vigna species for important agronomic traits.

  3. Sphingosine 1-phosphate elicits RhoA-dependent proliferation and MRTF-A mediated gene induction in CPCs.

    Castaldi, Alessandra; Chesini, Gino P; Taylor, Amy E; Sussman, Mark A; Brown, Joan Heller; Purcell, Nicole H


    Although c-kit(+) cardiac progenitor cells (CPCs) are currently used in clinical trials there remain considerable gaps in our understanding of the molecular mechanisms underlying their proliferation and differentiation. G-protein coupled receptors (GPCRs) play an important role in regulating these processes in mammalian cell types thus we assessed GPCR mRNA expression in c-kit(+) cells isolated from adult mouse hearts. Our data provide the first comprehensive overview of the distribution of this fundamental class of cardiac receptors in CPCs and reveal notable distinctions from that of adult cardiomyocytes. We focused on GPCRs that couple to RhoA activation in particular those for sphingosine-1-phosphate (S1P). The S1P2 and S1P3 receptors are the most abundant S1P receptor subtypes in mouse and human CPCs while cardiomyocytes express predominantly S1P1 receptors. Treatment of CPCs with S1P, as with thrombin and serum, increased proliferation through a pathway requiring RhoA signaling, as evidenced by significant attenuation when Rho was inhibited by treatment with C3 toxin. Further analysis demonstrated that both S1P- and serum-induced proliferation are regulated through the S1P2 and S1P3 receptor subtypes which couple to Gα12/13 to elicit RhoA activation. The transcriptional co-activator MRTF-A was activated by S1P as assessed by its nuclear accumulation and induction of a RhoA/MRTF-A luciferase reporter. In addition S1P treatment increased expression of cardiac lineage markers Mef2C and GATA4 and the smooth muscle marker GATA6 through activation of MRTF-A. In conclusion, we delineate an S1P-regulated signaling pathway in CPCs that introduces the possibility of targeting S1P2/3 receptors, Gα12/13 or RhoA to influence the proliferation and commitment of c-kit(+) CPCs and improve the response of the myocardium following injury.

  4. Evaluation of genetically modified sugarcane lines carrying Cry 1AC gene using molecular marker techniques.

    Ismail, Roba M


    Five genetically modified insect resistant sugarcane lines harboring the Bt Cry 1AC gene to produce insecticidal proteins were compared with non-transgenic control by using three types of molecular marker techniques namely, RAPD, ISSR and AFLP. These techniques were applied on transgenic and non-transgenic plants to investigate the genetic variations, which may appear in sugarcane clones. This variation might demonstrate the genomic changes associated with the transformation process, which could change important molecular basis of various biological phenomena. Genetic variations were screened using 22 different RAPD primers, 10 ISSR primers and 13 AFLP primer combinations. Analysis of RAPD and ISSR banding patterns gave no exclusive evidence for genetic variations. Meanwhile, the percentage of polymorphic bands was 0.45% in each of RAPD and ISSR, while the polymorphism generated by AFLP analysis was 1.8%. The maximum percentage of polymorphic bands was 1.4%, 1.1% and 5.5% in RAPD, ISSR and AFLP, respectively. These results demonstrate that most transgenic lines showed genomic homogeneity and verified minor genomic changes. Dendrograms revealing the relationships among the transgenic and control plants were developed from the data of each of the three marker types.

  5. Regulation of cell proliferation and apoptosis in neuroblastoma cells by ccp1, a FGF2 downstream gene

    Inman Gareth J


    Full Text Available Abstract Background Coiled-coil domain containing 115 (Ccdc115 or coiled coil protein-1 (ccp1 was previously identified as a downstream gene of Fibroblast Growth Factor 2 (FGF2 highly expressed in embryonic and adult brain. However, its function has not been characterised to date. Here we hypothesized that ccp1 may be a downstream effecter of FGF2, promoting cell proliferation and protecting from apoptosis. Methods Forced ccp1 expression in mouse embryonic fibroblast (MEF and neuroblastoma SK-N-SH cell line, as well as down-regulation of ccp1 expression by siRNA in NIH3T3, was used to characterize the role of ccp1. Results Ccp1 over-expression increased cell proliferation, whereas down-regulation of ccp1 expression reduced it. Ccp1 was able to increase cell proliferation in the absence of serum. Furthermore, ccp1 reduced apoptosis upon withdrawal of serum in SK-N-SH. The mitogen-activated protein kinase (MAPK or ERK Kinase (MEK inhibitor, U0126, only partially inhibited the ccp1-dependent BrdU incorporation, indicating that other signaling pathway may be involved in ccp1-induced cell proliferation. Induction of Sprouty (SPRY upon FGF2 treatment was accelerated in ccp1 over-expressing cells. Conclusions All together, the results showed that ccp1 regulates cell number by promoting proliferation and suppressing cell death. FGF2 was shown to enhance the effects of ccp1, however, it is likely that other mitogenic factors present in the serum can also enhance the effects. Whether these effects are mediated by FGF2 influencing the ccp1 function or by increasing the ccp1 expression level is still unclear. At least some of the proliferative regulation by ccp1 is mediated by MAPK, however other signaling pathways are likely to be involved.

  6. Expression of WNT genes in cervical cancer-derived cells: Implication of WNT7A in cell proliferation and migration

    Ramos-Solano, Moisés, E-mail: [División de Inmunología, Centro de Investigación Biomédica de Occidente (CIBO)-Instituto Mexicano del Seguro Social (IMSS), Guadalajara, Jalisco (Mexico); Programa de Doctorado en Ciencias Biomédica, Centro Universitario de Ciencias de la Salud (CUCS), Universidad de Guadalajara, Guadalajara, Jalisco (Mexico); Meza-Canales, Ivan D., E-mail: [Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, 07745 Jena (Germany); Torres-Reyes, Luis A., E-mail: [División de Inmunología, Centro de Investigación Biomédica de Occidente (CIBO)-Instituto Mexicano del Seguro Social (IMSS), Guadalajara, Jalisco (Mexico); Programa de Doctorado en Ciencias Biomédica, Centro Universitario de Ciencias de la Salud (CUCS), Universidad de Guadalajara, Guadalajara, Jalisco (Mexico); Alvarez-Zavala, Monserrat, E-mail: [División de Inmunología, Centro de Investigación Biomédica de Occidente (CIBO)-Instituto Mexicano del Seguro Social (IMSS), Guadalajara, Jalisco (Mexico); Programa de Doctorado en Ciencias Biomédica, Centro Universitario de Ciencias de la Salud (CUCS), Universidad de Guadalajara, Guadalajara, Jalisco (Mexico); and others


    According to the multifactorial model of cervical cancer (CC) causation, it is now recognized that other modifications, in addition to Human papillomavirus (HPV) infection, are necessary for the development of this neoplasia. Among these, it has been proposed that a dysregulation of the WNT pathway might favor malignant progression of HPV-immortalized keratinocytes. The aim of this study was to identify components of the WNT pathway differentially expressed in CC vs. non-tumorigenic, but immortalized human keratinocytes. Interestingly, WNT7A expression was found strongly downregulated in cell lines and biopsies derived from CC. Restoration of WNT7A in CC-derived cell lines using a lentiviral gene delivery system or after adding a recombinant human protein decreases cell proliferation. Likewise, WNT7A silencing in non-tumorigenic cells markedly accelerates proliferation. Decreased WNT7A expression was due to hypermethylation at particular CpG sites. To our knowledge, this is the first study reporting reduced WNT7A levels in CC-derived cells and that ectopic WNT7A restoration negatively affects cell proliferation and migration. - Highlights: • WNT7A is expressed in normal keratinocytes or cervical cells without lesion. • WNT7A is significantly reduced in cervical cancer-derived cells. • Restoration of WNT7A expression in HeLa decreases proliferation and cell migration. • Silencing of WNT7A in HaCaT induces an increased proliferation and migration rate. • Decreased WNT7A expression in this model is due to hypermethylation.

  7. [Detection of an NA gene molecular marker in H7N9 subtype avian influenza viruses by pyrosequencing].

    Zhao, Yong-Gang; Liu, Hua-Lei; Wang, Jing-Jing; Zheng, Dong-Xia; Zhao, Yun-Ling; Ge, Sheng-Qiang; Wang, Zhi-Liang


    This study aimed to establish a method for the detection and identification of H7N9 avian influenza viruses based on the NA gene by pyrosequencing. According to the published NA gene sequences of the avian influenza A (H7N9) virus, a 15-nt deletion was found in the NA gene of H7N9 avian influenza viruses. The 15-nt deletion of the NA gene was targeted as the molecular marker for the rapid detection and identification of H7N9 avian influenza viruses by pyrosequencing. Three H7N9 avian influenza virus isolates underwent pyrosequencing using the same assay, and were proven to have the same 15-nt deletion. Pyrosequencing technology based on the NA gene molecular marker can be used to identify H7N9 avian influenza viruses.

  8. HBV X-gene: A new serum marker for anti-HBV therapy monitoring


    Objective: To address HBV serum nucleic acid markers for stages without apparent replication. Methods: DNA and RNA sequence segments from the X, C and pre C/C regions produced successively during replication were used as targets for quantitative PCR and RT/PCR. Results: The assays confirmed the preferential formation of intermediates blocked at early stages. They persisted as the only detectable type of serum HBV DNA even after one year of therapy. At reentry into viral replication due to emergence of drug resistant mutants, lamivudine resistance produced exclusively incomplete DNA minus strands, whereas the wild type virus immediately synthesized complete DNA minus strands. Conclusion:PCR assays used for monitoring complete suppression of HBV replication must target the X gene region.

  9. Introgression of High Yield Genes from Lycopersicon hirsutum acc. LA1777 Using CAPS Marker

    LI Hong; WANG Xiao-xuan; SONG Ming; GAO Jian-chang; GUO Yan-mei; ZHU De-wei; DAI Shan-shu; DU Yong-chen


    The idea behind this study is to show that using high yield genes from a wild tomato can enrich tomato breeding resources and accelerate tomato breeding programs. In this study, the near-isogenic line TA1229 containing a 24-cM introgression at the bottom of chromosome 1 from Lycopersicon acc. LA1777, affects several higher yield traits. The TA1229 × 9706 BC1population was analyzed by marker-assisted selection and the traits of the population were evaluated. Twenty-three recombinant individuals that carried a shorter segment than TA1229 were obtained. Among them, 16 lines with the chromosome 1 recombinant segment can increase tomato yield and a QTL affecting yield was found between TG53 and TG158. Sixteen recombinant lines are useful to improve the tomato variety.

  10. Construction of interference vector targeting Ep-CAM gene and its effects on colorectal cancer cell proliferation.

    Qi, Yanmei; Zhou, Fengqiang; Zhang, Lu; Liu, Lei; Xu, Hong; Guo, Huiguang


    Prior study indicates that abnormal protein expression and functional changes in the development and progression of colorectal cancer is related to gene expression. The aim of this study was to construct an interference plasmid targeting the Ep-CAM gene and to investigate its effects on the proliferation of colorectal cancer cells. In this study, HT-29 and HCT-116 colorectal cancer cell lines were selected as cell models. The double-stranded micro (mi)RNA oligo was inserted into the pcDNATM6.2-GW/EmGFPmiR vector, which is an expression of miRNA. Lipofectamine™ 2000 was used to transfer plasmid into the empty plasmid group (transfected pcDNATM6.2-GW/EmGFPmiR-neg) and the interference group (transfected pcDNATM6.2-GW/EmGFPmiR-Ep-CAM-1), respectively. Meanwhile, the nontransferred HT-29 and HCT-116 acts as the blank control group. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the transfection efficiency. Western blot was used to detect Ep-CAM protein expression. The cell proliferation in each group was detected by using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results indicated that the Ep-CAM messenger (m)RNA expression in the interference group was lower significantly compared with that of the empty plasmid group and control group (PCAM protein expression was significantly lower in interference group compared with that of the empty plasmid group and the control group (Passay results demonstrated that the proliferation ability of cells in the interference group was significantly inhibited compared with the two other groups (PCAM can significantly inhibit the proliferation of colorectal cancer cells.

  11. Gene expression markers in circulating tumor cells may predict bone metastasis and response to hormonal treatment in breast cancer.

    Wang, Haiying; Molina, Julian; Jiang, John; Ferber, Matthew; Pruthi, Sandhya; Jatkoe, Timothy; Derecho, Carlo; Rajpurohit, Yashoda; Zheng, Jian; Wang, Yixin


    Circulating tumor cells (CTCs) have recently attracted attention due to their potential as prognostic and predictive markers for the clinical management of metastatic breast cancer patients. The isolation of CTCs from patients may enable the molecular characterization of these cells, which may help establish a minimally invasive assay for the prediction of metastasis and further optimization of treatment. Molecular markers of proven clinical value may therefore be useful in predicting disease aggressiveness and response to treatment. In our earlier study, we identified a gene signature in breast cancer that appears to be significantly associated with bone metastasis. Among the genes that constitute this signature, trefoil factor 1 (TFF1) was identified as the most differentially expressed gene associated with bone metastasis. In this study, we investigated 25 candidate gene markers in the CTCs of metastatic breast cancer patients with different metastatic sites. The panel of the 25 markers was investigated in 80 baseline samples (first blood draw of CTCs) and 30 follow-up samples. In addition, 40 healthy blood donors (HBDs) were analyzed as controls. The assay was performed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) with RNA extracted from CTCs captured by the CellSearch system. Our study indicated that 12 of the genes were uniquely expressed in CTCs and 10 were highly expressed in the CTCs obtained from patients compared to those obtained from HBDs. Among these genes, the expression of keratin 19 was highly correlated with the CTC count. The TFF1 expression in CTCs was a strong predictor of bone metastasis and the patients with a high expression of estrogen receptor β in CTCs exhibited a better response to hormonal treatment. Molecular characterization of these genes in CTCs may provide a better understanding of the mechanism underlying tumor metastasis and identify gene markers in CTCs for predicting disease progression and

  12. Disruption of polyubiquitin gene Ubc leads to defective proliferation of hepatocytes and bipotent fetal liver epithelial progenitor cells

    Park, Hyejin; Yoon, Min-Sik; Ryu, Kwon-Yul, E-mail:


    Highlights: •Proliferation capacity of Ubc{sup −/−} FLCs was reduced during culture in vitro. •Ubc is required for proliferation of both hepatocytes and bipotent FLEPCs. •Bipotent FLEPCs exhibit highest Ubc transcription and proliferation capacity. •Cell types responsible for Ubc{sup −/−} fetal liver developmental defect were identified. -- Abstract: We have previously demonstrated that disruption of polyubiquitin gene Ubc leads to mid-gestation embryonic lethality most likely due to a defect in fetal liver development, which can be partially rescued by ectopic expression of Ub. In a previous study, we assessed the cause of embryonic lethality with respect to the fetal liver hematopoietic system. We confirmed that Ubc{sup −/−} embryonic lethality could not be attributed to impaired function of hematopoietic stem cells, which raises the question of whether or not FLECs such as hepatocytes and bile duct cells, the most abundant cell types in the liver, are affected by disruption of Ubc and contribute to embryonic lethality. To answer this, we isolated FLCs from E13.5 embryos and cultured them in vitro. We found that proliferation capacity of Ubc{sup −/−} cells was significantly reduced compared to that of control cells, especially during the early culture period, however we did not observe the increased number of apoptotic cells. Furthermore, levels of Ub conjugate, but not free Ub, decreased upon disruption of Ubc expression in FLCs, and this could not be compensated for by upregulation of other poly- or mono-ubiquitin genes. Intriguingly, the highest Ubc expression levels throughout the entire culture period were observed in bipotent FLEPCs. Hepatocytes and bipotent FLEPCs were most affected by disruption of Ubc, resulting in defective proliferation as well as reduced cell numbers in vitro. These results suggest that defective proliferation of these cell types may contribute to severe reduction of fetal liver size and potentially mid

  13. Data on characterizing the gene expression patterns of neuronal ceroid lipofuscinosis genes: CLN1, CLN2, CLN3, CLN5 and their association to interneuron and neurotransmission markers: Parvalbumin and Somatostatin

    Helena M. Minye


    Full Text Available The article contains raw and analyzed data related to the research article “Neuronal ceroid lipofuscinosis genes, CLN2, CLN3, CLN5 are spatially and temporally co-expressed in a developing mouse brain” (Fabritius et al., 2014 [1]. The processed data gives an understanding of the development of the cell types that are mostly affected by defective function of CLN proteins, timing of expression of CLN1, CLN2, CLN3 and CLN5 genes in a murine model. The data shows relationship between the expression pattern of these genes during neural development. Immunohistochemistry was used to identify known interneuronal markers for neurotransmission and cell proliferation: parvalbumin, somatostatin subpopulations of interneurons. Non-radioactive in-situ hybridization detected CLN5 mRNA in the hippocampus. Throughout the development strong expression of CLN genes were identified in the germinal epithelium and in ventricle regions, cortex, hippocampus, and cerebellum. This provides supportive evidence that CLN1, CLN2, CLN3 and CLN5 genes may be involved in synaptic pruning.

  14. Gene amplification and immunohistochemical expression of ERBB2 and EGFR in cervical carcinogenesis. Correlation with cell-cycle markers and HPV presence.

    Conesa-Zamora, Pablo; Torres-Moreno, Daniel; Isaac, María A; Pérez-Guillermo, Miguel


    Although the members of the epidermal growth factor receptor family ERBB2 and EGFR are important therapeutic targets in the treatment of malignant neoplasias, little is known about their role in cervical carcinogenesis. Our objective was to evaluate the dysfunction of ERBB2 and EGFR at the gene copy number and protein expression level in neoplastic lesions of the uterine cervix with the aim of obtaining information about its role in cervical carcinogenesis and their possible use as therapeutic targets in these diseases. We studied gene amplification and protein expression of ERBB2 and EGFR and their relationship with Ki67, p16 and p53 and HPV presence in 22 normal/benign (N/B) cervices, 20 low-grade squamous intraepithelial lesions (LSILs), 70 high-grade SILs (HSILs) and 32 invasive squamous cervical carcinomas (ISCCs). No cases showed selective amplification of ERBB2 or EGFR but corresponding chromosome-specific probes displayed chromosome 17 and 7 polyploidy associated with the grade of the lesion (plesions (N/B plus LSIL 21.4% vs. HSIL plus ISCC 45.5%; p=0.007). No association was observed between EGFR expression and that of cell-cycle markers or HPV presence. Increased copy number of EGFR and ERBB2 is due to polyploidy of 7 and 17 chromosomes, this being a phenomenon associated with lesion severity and with an increase in the expression of cell-cycle markers. EGFR, but not ERBB2, is expressed in precursor lesions of squamous cervical neoplasia and is related to the neoplastic progression but not to proliferation marker expression and therefore ERBB2 and this calls into question the usefulness of ERBB2 as a therapeutic target.

  15. EGFR/Ras Signaling Controls Drosophila Intestinal Stem Cell Proliferation via Capicua-Regulated Genes

    Jin, Yinhua; Ha, Nati; Forés, Marta; Xiang, Jinyi; Gläßer, Christine; Maldera, Julieta; Jiménez, Gerardo; Edgar, Bruce A.


    Epithelial renewal in the Drosophila intestine is orchestrated by Intestinal Stem Cells (ISCs). Following damage or stress the intestinal epithelium produces ligands that activate the epidermal growth factor receptor (EGFR) in ISCs. This promotes their growth and division and, thereby, epithelial regeneration. Here we demonstrate that the HMG-box transcriptional repressor, Capicua (Cic), mediates these functions of EGFR signaling. Depleting Cic in ISCs activated them for division, whereas overexpressed Cic inhibited ISC proliferation and midgut regeneration. Epistasis tests showed that Cic acted as an essential downstream effector of EGFR/Ras signaling, and immunofluorescence showed that Cic’s nuclear localization was regulated by EGFR signaling. ISC-specific mRNA expression profiling and DNA binding mapping using DamID indicated that Cic represses cell proliferation via direct targets including string (Cdc25), Cyclin E, and the ETS domain transcription factors Ets21C and Pointed (pnt). pnt was required for ISC over-proliferation following Cic depletion, and ectopic pnt restored ISC proliferation even in the presence of overexpressed dominant-active Cic. These studies identify Cic, Pnt, and Ets21C as critical downstream effectors of EGFR signaling in Drosophila ISCs. PMID:26683696

  16. BABY BOOM target genes provide diverse entry points into cell proliferation and cell growth pathways

    Passarinho, P.A.; Ketelaar, M.J.; Xing, M.; Arkel, van J.; Maliepaard, C.A.; Weemen, W.M.J.; Joosen, R.V.L.; Lammers, M.; Herdies, L.; Boer, de B.; Geest, van der A.H.M.; Boutilier, K.A.


    Ectopic expression of the Brassica napus BABY BOOM (BBM) AP2/ERF transcription factor is sufficient to induce spontaneous cell proliferation leading primarily to somatic embryogenesis, but also to organogenesis and callus formation. We used DNA microarray analysis in combination with a

  17. Effects of PCBs and PBDEs on thyroid hormone, lymphocyte proliferation, hematology and kidney injury markers in residents of an e-waste dismantling area in Zhejiang, China

    Xu, Peiwei, E-mail:; Lou, Xiaoming; Ding, Gangqiang, E-mail:; Shen, Haitao; Wu, Lizhi; Chen, Zhijian; Han, Jianlong; Wang, Xiaofeng, E-mail:


    Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are two typical categories of contaminants released from e-waste dismantling environments. In China, the body burdens of PCBs and PBDEs are associated with abnormal thyroid hormones in populations from e-waste dismantling sites, but the results are limited and contradictory. In this study, we measured the serum levels of PCBs and PBDEs and the thyroid hormone free triiodothyronine (FT3), free thyroxine (FT4) and thyroid-stimulating hormone (TSH) in 40 residents in an e-waste dismantling area and in 15 residents in a control area. Additionally, we also measured some lymphocyte proliferation indexes, hematologic parameters and kidney injury markers, including white blood cells, neutrophils, monocytes, lymphocytes, hemoglobin, platelets, serum creatinine and beta 2-microglobulin (β{sub 2}-MG). The results indicated that the mean level of ΣPCBs in the exposure group was significantly higher than that in the control group (964.39 and 67.98 ng g{sup −1}, p < 0.0001), but the mean level of ΣPBDEs in the exposure group was not significantly higher than that in the controls (139.32 vs. 75.74 ng g{sup −1}, p > 0.05). We determined that serum levels of FT3, FT4, monocytes and lymphocytes were significantly lower, whereas the levels of neutrophils, hemoglobin, platelets and serum creatinine were significantly higher in the exposed group (p < 0.05). The mean level of ΣPCBs was negatively correlated with levels of FT3, FT4, monocytes and lymphocytes (p < 0.05) and positively correlated with levels of neutrophils, hemoglobin, serum creatinine and β{sub 2}-MG (p < 0.05). Additionally, the mean level of ΣPBDEs was positively correlated with levels of white blood cells, hemoglobin and platelets (p < 0.05). Our data suggest that exposure to an e-waste dismantling environment may increase the body burdens of PCBs and the specific PBDEs congeners in native residents and that the contaminants released

  18. Minimum information about a marker gene sequence (MIMARKS) and minimum information about any (x) sequence (MIxS) specifications

    Yilmaz, Pelin; Kottmann, Renzo; Field, Dawn


    Here we present a standard developed by the Genomic Standards Consortium (GSC) for reporting marker gene sequences--the minimum information about a marker gene sequence (MIMARKS). We also introduce a system for describing the environment from which a biological sample originates. The 'environmental...... present the minimum information about any (x) sequence (MIxS). Adoption of MIxS will enhance our ability to analyze natural genetic diversity documented by massive DNA sequencing efforts from myriad ecosystems in our ever-changing biosphere....

  19. Long non-coding RNA small nucleolar RNA host gene 12 (SNHG12) promotes cell proliferation and migration by upregulating angiomotin gene expression in human osteosarcoma cells.

    Ruan, Wendong; Wang, Pei; Feng, Shiqing; Xue, Yuan; Li, Yulin


    The long non-coding RNA (lncRNA) small nucleolar RNA host gene 12 (SNHG12) has a role in cell proliferation and migration. Angiomotin, encoded by the AMOT gene, is a protein that regulates the migration and organization of endothelial cells. SNHG12 and AMOT have been shown to play a role in a variety of human cancers but have yet to be studied in detail in human osteosarcoma. Tissue samples from primary osteosarcoma (n = 20) and adjacent normal tissues (n = 20), the osteosarcoma cell lines, SAOS-2, MG-63, U-2 OS, and the human osteoblast cell line hFOB (OB3) were studied using Western blot for angiomotin, and quantitative real-time polymerase chain reaction for the expression of SNHG12 and AMOT. The expression of SNHG12 was knocked down using RNA interference. Cell migration assays were performed. Cell apoptosis was studied using flow cytometry. SNHG12 and AMOT messenger RNA (mRNA) expression was upregulated in osteosarcoma tissues and cell lines when compared with normal tissues and cells. Upregulation of AMOT mRNA was associated with upregulation of SNHG12. Knockdown of SNHG12 reduced the expression of angiomotin in osteosarcoma cells and suppressed cell proliferation and migration but did not affect cell apoptosis. This preliminary study has shown that the lncRNA SNHG12 promotes cell proliferation and migration by upregulating AMOT gene expression in osteosarcoma cells in vivo and in vitro. Further studies are recommended to investigate the role of SNHG12 and AMOT expression in tumor cell proliferation and migration and angiogenesis in osteosarcoma and a range of malignant mesenchymal tumors.

  20. Effects of different doses of 2-methoxy-estradiol on the proliferation, apoptosis and angiogenesis genes in malignant melanoma cells

    Xiao-Bo Tong


    Objective:To study the inhibitory effect of different doses of 2-methoxy-estradiol on the growth of malignant melanoma cells in vitro.Methods:First, melanoma B16 cells were cultured, and then 0μmol / L, 10 μmol / L, 20 μmol / L, 30umol / L and 40 umol / L of 2-ME were added. Last, cell viability was detected MTS kit, and the contents of proliferation gene, apoptosis gene and angiogenesis gene in both cells and culture medium were determined by Elisa.Results:2-ME reduced cell viability in a dose-dependent and time-dependent way. After 40 umol/L of 2-ME treatment, Mcl-1 and CYR61 contents in cells decreased significantly, while Fas and Caspase14 contents increased significantly. HIF-1α, VEGF, SDF-1 and CXCR4 decreased significantly in both cells and culture medium.Conclusions:Different doses of 2-ME can inhibit the growth of malignant melanoma cells in vitro by reducing the cell viability and inhibiting cell proliferation and angiogenesis.

  1. Suppression of cell division-associated genes by Helicobacter pylori attenuates proliferation of RAW264.7 monocytic macrophage cells.

    Tan, Grace Min Yi; Looi, Chung Yeng; Fernandez, Keith Conrad; Vadivelu, Jamuna; Loke, Mun Fai; Wong, Won Fen


    Helicobacter pylori at multiplicity of infection (MOI ≥ 50) have been shown to cause apoptosis in RAW264.7 monocytic macrophage cells. Because chronic gastric infection by H. pylori results in the persistence of macrophages in the host's gut, it is likely that H. pylori is present at low to moderate, rather than high numbers in the infected host. At present, the effect of low-MOI H. pylori infection on macrophage has not been fully elucidated. In this study, we investigated the genome-wide transcriptional regulation of H. pylori-infected RAW264.7 cells at MOI 1, 5 and 10 in the absence of cellular apoptosis. Microarray data revealed up- and down-regulation of 1341 and 1591 genes, respectively. The expression of genes encoding for DNA replication and cell cycle-associated molecules, including Aurora-B kinase (AurkB) were down-regulated. Immunoblot analysis verified the decreased expression of AurkB and downstream phosphorylation of Cdk1 caused by H. pylori infection. Consistently, we observed that H. pylori infection inhibited cell proliferation and progression through the G1/S and G2/M checkpoints. In summary, we suggest that H. pylori disrupts expression of cell cycle-associated genes, thereby impeding proliferation of RAW264.7 cells, and such disruption may be an immunoevasive strategy utilized by H. pylori.

  2. Proliferation and C-myc Gene Expression of Smooth Muscle Cells in Rabbit Carotid Artery after Stenting

    张新霞; 崔长琮; 胡雪松; 魏文斌; 李松; 许香广


    Objectives To investigate the proliferation of smooth muscle cells (VSMCs) and the expression of c-myc gene in rabbit carotid arteries after stenting. Methods Platinium-Iridium stent were implanted into the right carotid arteries of 16 rabbits under vision. 7,14,30 and 90 days after the stenting procedure, morphological changes of VSMCs were observed under light and transmission electron microscope. The c-myc gene expression was detected by in situ hybridization (ISH) and immunohistochemical staining. Results 7 days after stenting, the phenotype of VSMCs changed from contractile to synthetic phenotype; there were a number of proliferative VSMCs in the neointima. At 14 and 30 days, there were synthetic and transitive VSMCs. At 90 days, the phenotype of VSMCs recovered to contractile phenotype.The ultrastructure of typical synthetic phenotype of VSMCs were round, containing a large amount of rough endoplasmic reticulum and mitochondria. Cmyc expression were positive both by ISH and immunohistochemical staining. Conclusions C-myc gene expression increases and closely relates to VSMCs proliferation after stenting. It may play an important role in the in-stent restenosis.

  3. Nitric Oxide Prevents Mouse Embryonic Stem Cell Differentiation Through Regulation of Gene Expression, Cell Signaling, and Control of Cell Proliferation.

    Tapia-Limonchi, Rafael; Cahuana, Gladys M; Caballano-Infantes, Estefania; Salguero-Aranda, Carmen; Beltran-Povea, Amparo; Hitos, Ana B; Hmadcha, Abdelkrim; Martin, Franz; Soria, Bernat; Bedoya, Francisco J; Tejedo, Juan R


    Nitric oxide (NO) delays mouse embryonic stem cell (mESC) differentiation by regulating genes linked to pluripotency and differentiation. Nevertheless, no profound study has been conducted on cell differentiation regulation by this molecule through signaling on essential biological functions. We sought to demonstrate that NO positively regulates the pluripotency transcriptional core, enforcing changes in the chromatin structure, in addition to regulating cell proliferation, and signaling pathways with key roles in stemness. Culturing mESCs with 2 μM of the NO donor diethylenetriamine/NO (DETA/NO) in the absence of leukemia inhibitory factor (LIF) induced significant changes in the expression of 16 genes of the pluripotency transcriptional core. Furthermore, treatment with DETA/NO resulted in a high occupancy of activating H3K4me3 at the Oct4 and Nanog promoters and repressive H3K9me3 and H3k27me3 at the Brachyury promoter. Additionally, the activation of signaling pathways involved in pluripotency, such as Gsk3-β/β-catenin, was observed, in addition to activation of PI3 K/Akt, which is consistent with the protection of mESCs from cell death. Finally, a decrease in cell proliferation coincides with cell cycle arrest in G2/M. Our results provide novel insights into NO-mediated gene regulation and cell proliferation and suggest that NO is necessary but not sufficient for the maintenance of pluripotency and the prevention of cell differentiation. J. Cell. Biochem. 117: 2078-2088, 2016. © 2016 Wiley Periodicals, Inc.

  4. Identification of miR-2400 gene as a novel regulator in skeletal muscle satellite cells proliferation by targeting MYOG gene

    Zhang, Wei Wei [The Laboratory of Cell and Development, Northeast Agricultural University, Harbin, Heilongjiang 150030 (China); College of Life Sciences and Agriculture & Forestry, Qiqihar University, Qiqihar, Heilongjiang 161006 (China); Tong, Hui Li; Sun, Xiao Feng; Hu, Qian; Yang, Yu; Li, Shu Feng [The Laboratory of Cell and Development, Northeast Agricultural University, Harbin, Heilongjiang 150030 (China); Yan, Yun Qin, E-mail: [The Laboratory of Cell and Development, Northeast Agricultural University, Harbin, Heilongjiang 150030 (China); Li, Guang Peng [The Key Laboratory of Mammal Reproductive Biology and Biotechnology Ministry of Education, Inner Mongolia University, Hohhot 010021 (China)


    MicroRNAs play critical roles in skeletal muscle development as well as in regulation of muscle cell proliferation and differentiation. Previous study in our laboratory showed that the expression level of miR-2400, a novel and unique miRNA from bovine, had significantly changed in skeletal muscle-derived satellite cells (MDSCs) during differentiation, however, the function and expression pattern for miR-2400 in MDSCs has not been fully understood. In this report, we firstly identified that the expression levels of miR-2400 were down-regulated during MDSCs differentiation by stem-loop RT-PCR. Over-expression and inhibition studies demonstrated that miR-2400 promoted MDSCs proliferation by EdU (5-ethynyl-2′ deoxyuridine) incorporation assay and immunofluorescence staining of Proliferating cell nuclear antigen (PCNA). Luciferase reporter assays showed that miR-2400 directly targeted the 3′ untranslated regions (UTRs) of myogenin (MYOG) mRNA. These data suggested that miR-2400 could promote MDSCs proliferation through targeting MYOG. Furthermore, we found that miR-2400, which was located within the eighth intron of the Wolf-Hirschhorn syndrome candidate 1-like 1 (WHSC1L1) gene, was down-regulated in MDSCs in a direct correlation with the WHSC1L1 transcript by Clustered regularly interspaced palindromic repeats interference (CRISPRi). In addition, these observations not only provided supporting evidence for the codependent expression of intronic miRNAs and their host genes in vitro, but also gave insight into the role of miR-2400 in MDSCs proliferation. - Highlights: • miR-2400 is a novel and unique miRNA from bovine. • miR-2400 could promote skeletal muscle satellite cells proliferation. • miR-2400 directly targeted the 3′ untranslated regions of MYOG mRNA. • miR-2400 could be coexpressed together with its host gene WHSC1L1.

  5. Pituitary adenylyl cyclase activating polypeptide inhibits gli1 gene expression and proliferation in primary medulloblastoma derived tumorsphere cultures

    Dong Hongmei


    Full Text Available Abstract Background Hedgehog (HH signaling is critical for the expansion of granule neuron precursors (GNPs within the external granular layer (EGL during cerebellar development. Aberrant HH signaling within GNPs is thought to give rise to medulloblastoma (MB - the most commonly-observed form of malignant pediatric brain tumor. Evidence in both invertebrates and vertebrates indicates that cyclic AMP-dependent protein kinase A (PKA antagonizes HH signalling. Receptors specific for the neuropeptide pituitary adenylyl cyclase activating polypeptide (PACAP, gene name ADCYAP1 are expressed in GNPs. PACAP has been shown to protect GNPs from apoptosis in vitro, and to interact with HH signaling to regulate GNP proliferation. PACAP/ptch1 double mutant mice exhibit an increased incidence of MB compared to ptch1 mice, indicating that PACAP may regulate HH pathway-mediated MB pathogenesis. Methods Primary MB tumorsphere cultures were prepared from thirteen ptch1+/-/p53+/- double mutant mice and treated with the smoothened (SMO agonist purmorphamine, the SMO antagonist SANT-1, the neuropeptide PACAP, the PKA activator forskolin, and the PKA inhibitor H89. Gene expression of gli1 and [3H]-thymidine incorporation were assessed to determine drug effects on HH pathway activity and proliferation, respectively. PKA activity was determined in cell extracts by Western blotting using a phospho-PKA substrate antibody. Results Primary tumor cells cultured for 1-week under serum-free conditions grew as tumorspheres and were found to express PAC1 receptor transcripts. Gli1 gene expression was significantly reduced by SANT-1, PACAP and forskolin, but was unaffected by purmorphamine. The attenuation of gli1 gene expression by PACAP was reversed by the PKA inhibitor H89, which also blocked PKA activation. Treatment of tumorsphere cultures with PACAP, forskolin, and SANT-1 for 24 or 48 hours reduced proliferation. Conclusions Primary tumorspheres derived from ptch1+/-/p53

  6. Expression of a Buckwheat Trypsin Inhibitor Gene in Escherichia coli and its Effect on Multiple Myeloma IM-9 Cell Proliferation


    The gene of buckwheat trypsin inhibitor (BTI) has been cloned and expressed in Escherichia coli. The yield of this recombinant inhibitor was over 12 mg/L by using one-step purification on a Ni2+-NTA Sepharose column. Its molecular weight was 9322.1 Da, determined by mass spectrum analysis. The MTT and cytometry analyses showed that recombinant BTI could specifically inhibit the proliferation of IM-9 human B lymphoblastoid cells (from patient with multiple myeloma) in a dose-dependent manner. The test of recombinant BTI-induced apoptosis in IM-9 cells implied that the inhibitor might have potential application in the treatment of cancer.

  7. A new marker based on the avian spindlin gene that is able to sex most birds, including species problematic to sex with CHD markers.

    Dawson, Deborah A; Dos Remedios, Natalie; Horsburgh, Gavin J


    We have developed a new marker (Z43B) that can be successfully used to identify the sex of most birds (69%), including species difficult or impossible to sex with other markers. We utilized the zebra finch Taeniopygia guttata EST microsatellite sequence (CK309496) which displays sequence homology to the 5' untranslated region (UTR) of the avian spindlin gene. This gene is known to be present on the Z and W chromosomes. To maximize cross-species utility, the primer set was designed from a consensus sequence created from homologs of CK309496 that were isolated from multiple distantly related species. Both the forward and reverse primer sequences were 100% identical to 14 avian species, including the Z chromosome of eight species and the chicken Gallus gallus W chromosome, as well as the saltwater crocodile Crocodylus porosus. The Z43B primer set was assessed by genotyping individuals of known sex belonging to 61 non-ratite species and a single ratite. The Z and W amplicons differed in size making it possible to distinguish between males (ZZ) and females (ZW) for the majority (69%) of non-ratite species tested, comprising 10 orders of birds. We predict that this marker will be useful for obtaining sex-typing data for ca 6,869 species of birds (69% of non-ratites but not galliforms). A wide range of species could be sex-typed including passerines, shorebirds, eagles, falcons, bee-eaters, cranes, shags, parrots, penguins, ducks, and a ratite species, the brown kiwi, Apteryx australis. Those species sexed include species impossible or problematic to sex-type with other markers (magpie, albatross, petrel, eagle, falcon, crane, and penguin species). Zoo Biol. 35:533-545, 2016. © 2016 The Authors. Zoo Biology published by Wiley Periodicals, Inc. © 2016 The Authors. Zoo Biology published by Wiley Periodicals, Inc.

  8. New mucin-like cancer-associated antigens (CA M 26, CA M 29 and CA 549) and a new proliferation marker (TPS) in patients with primary or advanced breast cancer.

    Locker, G J; Mader, R M; Braun, J; Sieder, A E; Marosi, C; Rainer, H; Jakesz, R; Steger, G G


    In patients with breast cancer no tumor markers giving satisfactory results have been found yet. The aim of our investigation was to compare the usefulness of newly developed tumor markers with the most common used carcinoembryonic antigen and cancer antigen (CA) 15-3. We evaluated the concentrations of carcinoma-associated antigen (CA) 549, carcinoma-associated mucin antigen (CA M) 26 and CA M 29, and the proliferation markers tissue polypeptide antigen (TPA) and tissue polypeptide-specific antigen (TPS) in 84 breast cancer patients with disease progression and in 69 patients with no evidence of disease after surgery for breast cancer. Using receiver-operating characteristic curves (ROC curves) we were able to demonstrate increased sensitivity and specificity of all tested tumor markers in patients with metastatic disease compared with local disease. In our investigation TPA is superior to TPS in all disease states. In local disease, none of the tested markers shows satisfying results. In metastatic disease, the new mucin markers CA M 26 and CA M 29 show slightly better results than CA 15-3 although their ROC curves are nearly congruent. CA 549 is exceeded by the other mucin markers. The best results in this investigation were obtained with CA M 29. The overall results concerning the detection of small tumor masses (i.e. local disease) were unsatisfactory.

  9. HPV16 E2 could act as down-regulator in cellular genes implicated in apoptosis, proliferation and cell differentiation

    Valencia-Hernández Armando


    Full Text Available Abstract Background Human Papillomavirus (HPV E2 plays several important roles in the viral cycle, including the transcriptional regulation of the oncogenes E6 and E7, the regulation of the viral genome replication by its association with E1 helicase and participates in the viral genome segregation during mitosis by its association with the cellular protein Brd4. It has been shown that E2 protein can regulate negative or positively the activity of several cellular promoters, although the precise mechanism of this regulation is uncertain. In this work we constructed a recombinant adenoviral vector to overexpress HPV16 E2 and evaluated the global pattern of biological processes regulated by E2 using microarrays expression analysis. Results The gene expression profile was strongly modified in cells expressing HPV16 E2, finding 1048 down-regulated genes, and 581 up-regulated. The main cellular pathway modified was WNT since we found 28 genes down-regulated and 15 up-regulated. Interestingly, this pathway is a convergence point for regulating the expression of genes involved in several cellular processes, including apoptosis, proliferation and cell differentiation; MYCN, JAG1 and MAPK13 genes were selected to validate by RT-qPCR the microarray data as these genes in an altered level of expression, modify very important cellular processes. Additionally, we found that a large number of genes from pathways such as PDGF, angiogenesis and cytokines and chemokines mediated inflammation, were also modified in their expression. Conclusions Our results demonstrate that HPV16 E2 has regulatory effects on cellular gene expression in HPV negative cells, independent of the other HPV proteins, and the gene profile observed indicates that these effects could be mediated by interactions with cellular proteins. The cellular processes affected suggest that E2 expression leads to the cells in to a convenient environment for a replicative cycle of the virus.

  10. Gene-associated markers provide tools for tackling illegal fishing and false eco-certification.

    Nielsen, Einar E; Cariani, Alessia; Mac Aoidh, Eoin; Maes, Gregory E; Milano, Ilaria; Ogden, Rob; Taylor, Martin; Hemmer-Hansen, Jakob; Babbucci, Massimiliano; Bargelloni, Luca; Bekkevold, Dorte; Diopere, Eveline; Grenfell, Leonie; Helyar, Sarah; Limborg, Morten T; Martinsohn, Jann T; McEwing, Ross; Panitz, Frank; Patarnello, Tomaso; Tinti, Fausto; Van Houdt, Jeroen K J; Volckaert, Filip A M; Waples, Robin S; Albin, Jan E J; Vieites Baptista, Juan M; Barmintsev, Vladimir; Bautista, José M; Bendixen, Christian; Bergé, Jean-Pascal; Blohm, Dietmar; Cardazzo, Barbara; Diez, Amalia; Espiñeira, Montserrat; Geffen, Audrey J; Gonzalez, Elena; González-Lavín, Nerea; Guarniero, Ilaria; Jeráme, Marc; Kochzius, Marc; Krey, Grigorius; Mouchel, Olivier; Negrisolo, Enrico; Piccinetti, Corrado; Puyet, Antonio; Rastorguev, Sergey; Smith, Jane P; Trentini, Massimo; Verrez-Bagnis, Véronique; Volkov, Alexander; Zanzi, Antonella; Carvalho, Gary R


    Illegal, Unreported and Unregulated fishing has had a major role in the overexploitation of global fish populations. In response, international regulations have been imposed and many fisheries have been 'eco-certified' by consumer organizations, but methods for independent control of catch certificates and eco-labels are urgently needed. Here we show that, by using gene-associated single nucleotide polymorphisms, individual marine fish can be assigned back to population of origin with unprecedented high levels of precision. By applying high differentiation single nucleotide polymorphism assays, in four commercial marine fish, on a pan-European scale, we find 93-100% of individuals could be correctly assigned to origin in policy-driven case studies. We show how case-targeted single nucleotide polymorphism assays can be created and forensically validated, using a centrally maintained and publicly available database. Our results demonstrate how application of gene-associated markers will likely revolutionize origin assignment and become highly valuable tools for fighting illegal fishing and mislabelling worldwide.

  11. Antibiotic resistance marker genes as environmental pollutants in GMO-pristine agricultural soils in Austria.

    Woegerbauer, Markus; Zeinzinger, Josef; Gottsberger, Richard Alexander; Pascher, Kathrin; Hufnagl, Peter; Indra, Alexander; Fuchs, Reinhard; Hofrichter, Johannes; Kopacka, Ian; Korschineck, Irina; Schleicher, Corina; Schwarz, Michael; Steinwider, Johann; Springer, Burkhard; Allerberger, Franz; Nielsen, Kaare M; Fuchs, Klemens


    Antibiotic resistance genes may be considered as environmental pollutants if anthropogenic emission and manipulations increase their prevalence above usually occurring background levels. The prevalence of aph(3')-IIa/nptII and aph(3')-IIIa/nptIII - frequent marker genes in plant biotechnology conferring resistance to certain aminoglycosides - was determined in Austrian soils from 100 maize and potato fields not yet exposed to but eligible for GMO crop cultivation. Total soil DNA extracts were analysed by nptII/nptIII-specific TaqMan real time PCR. Of all fields 6% were positive for nptII (median: 150 copies/g soil; range: 31-856) and 85% for nptIII (1190 copies/g soil; 13-61600). The copy-number deduced prevalence of nptIII carriers was 14-fold higher compared to nptII. Of the cultivable kanamycin-resistant soil bacteria 1.8% (95% confidence interval: 0-3.3%) were positive for nptIII, none for nptII (0-0.8%). The nptII-load of the studied soils was low rendering nptII a typical candidate as environmental pollutant upon anthropogenic release into these ecosystems. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Construction of Antisense Transforming Growth Factorβ1 Gene and Its Effect on the Proliferation by Expression in Osteosarcoma Cells

    刘勇; 郑启新; 杜靖远; 杨述华; 邵增务; 肖宝钧


    Summary: To construct the antisensc transforming growth factorβl (TGFβ1) gene and investigatethe effect of TGFβ1 autocrine loop blockage on the proliferation of osteosarcoma cells. TGFβ1 cDNAwas cloned by RT-PCR from human osteosarcoma cells (MG-63) and inserted into pcDNA3 to con-struct an antisense expression vector, which was dubbed pcDNA3-TGFβ1(- ). MTT was used to de-tect the proliferation of osteosarcoma cells transfected by antisense TGFβ1 gene. Our results showedthat the proliferation of the transfected osteosarcoma cells was suppressed markedly. It is concludedthat TGFβ1 autocrine loop blockage in osteosarcoma cells could inhibit cell proliferation, which mightbe helpful for gene therapy of osteosarcoma.

  13. Codominant PCR-based markers and candidate genes for powdery mildew resistance in melon (Cucumis melo L.).

    Yuste-Lisbona, Fernando J; Capel, Carmen; Gómez-Guillamón, María L; Capel, Juan; López-Sesé, Ana I; Lozano, Rafael


    Powdery mildew caused by Podosphaera xanthii is a major disease in melon crops, and races 1, 2, and 5 of this fungus are those that occur most frequently in southern Europe. The genotype TGR-1551 bears a dominant gene that provides resistance to these three races of P. xanthii. By combining bulked segregant analysis and amplified fragment length polymorphisms (AFLP), we identified eight markers linked to this dominant gene. Cloning and sequencing of the selected AFLP fragments allowed the development of six codominant PCR-based markers which mapped on the linkage group (LG) V. Sequence analysis of these markers led to the identification of two resistance-like genes, MRGH5 and MRGH63, belonging to the nucleotide binding site (NBS)-leucine-rich repeat (LRR) gene family. Quantitative trait loci (QTL) analysis detected two QTLs, Pm-R1-2 and Pm-R5, the former significantly associated with the resistance to races 1 and 2 (LOD score of 26.5 and 33.3; 53.6 and 61.9% of phenotypic variation, respectively), and the latter with resistance to race 5 (LOD score of 36.8; 65.5% of phenotypic variation), which have been found to be colocalized with the MRGH5 and MRGH63 genes, respectively. The results suggest that the cluster of NBS-LRR genes identified in LG V harbours candidate genes for resistance to races 1, 2, and 5 of P. xanthii. The evaluation of other resistant germplasm showed that the codominant markers here reported are also linked to the Pm-w resistance gene carried by the accession 'WMR-29' proving their usefulness as genotyping tools in melon breeding programmes.

  14. Genotype-Based Bayesian Analysis of Gene-Environment Interactions with Multiple Genetic Markers and Misclassification in Environmental Factors

    Iryna Lobach; Ruzong Fan


    A key component to understanding etiology of complex diseases, such as cancer, diabetes, alcohol dependence, is to investigate gene-environment interactions. This work is motivated by the following two concerns in the analysis of gene-environment interactions. First, multiple genetic markers in moderate linkage disequilibrium may be involved in susceptibility to a complex disease. Second, environmental factors may be subject to misclassification. We develop a genotype based Bayesian pseudolik...

  15. Microglial CD206 Gene Has Potential as a State Marker of Bipolar Disorder

    Ohgidani, Masahiro; Kato, Takahiro A.; Haraguchi, Yoshinori; Matsushima, Toshio; Mizoguchi, Yoshito; Murakawa-Hirachi, Toru; Sagata, Noriaki; Monji, Akira; Kanba, Shigenobu


    The pathophysiology of bipolar disorder, especially the underlying mechanisms of the bipolarity between manic and depressive states, has yet to be clarified. Microglia, immune cells in the brain, play important roles in the process of brain inflammation, and recent positron emission tomography studies have indicated microglial overactivation in the brain of patients with bipolar disorder. We have recently developed a technique to induced microglia-like (iMG) cells from peripheral blood (monocytes). We introduce a novel translational approach focusing on bipolar disorder using this iMG technique. We hypothesize that immunological conditional changes in microglia may contribute to the shift between manic and depressive states, and thus we herein analyzed gene profiling patterns of iMG cells from three patients with rapid cycling bipolar disorder during both manic and depressive states, respectively. We revealed that the gene profiling patterns are different between manic and depressive states. The profiling pattern of case 1 showed that M1 microglia is dominant in the manic state compared to the depressive state. However, the patterns of cases 2 and 3 were not consistent with the pattern of case 1. CD206, a mannose receptor known as a typical M2 marker, was significantly downregulated in the manic state among all three patients. This is the first report to indicate the importance of shifting microglial M1/M2 characteristics, especially the CD206 gene expression pattern between depressive and manic states. Further translational studies are needed to dig up the microglial roles in the underlying biological mechanisms of bipolar disorder. PMID:28119691

  16. Meta-analysis of several gene lists for distinct types of cancer: A simple way to reveal common prognostic markers

    Sun Xiao


    Full Text Available Abstract Background Although prognostic biomarkers specific for particular cancers have been discovered, microarray analysis of gene expression profiles, supported by integrative analysis algorithms, helps to identify common factors in molecular oncology. Similarities of Ordered Gene Lists (SOGL is a recently proposed approach to meta-analysis suitable for identifying features shared by two data sets. Here we extend the idea of SOGL to the detection of significant prognostic marker genes from microarrays of multiple data sets. Three data sets for leukemia and the other six for different solid tumors are used to demonstrate our method, using established statistical techniques. Results We describe a set of significantly similar ordered gene lists, representing outcome comparisons for distinct types of cancer. This kind of similarity could improve the diagnostic accuracies of individual studies when SOGL is incorporated into the support vector machine algorithm. In particular, we investigate the similarities among three ordered gene lists pertaining to mesothelioma survival, prostate recurrence and glioma survival. The similarity-driving genes are related to the outcomes of patients with lung cancer with a hazard ratio of 4.47 (p = 0.035. Many of these genes are involved in breakdown of EMC proteins regulating angiogenesis, and may be used for further research on prognostic markers and molecular targets of gene therapy for cancers. Conclusion The proposed method and its application show the potential of such meta-analyses in clinical studies of gene expression profiles.

  17. Analysis of environmental stress in plants with the aid of marker genes for H2O2 responses.

    Hieno, Ayaka; Naznin, Hushna Ara; Sawaki, Katsunobu; Koyama, Hiroyuki; Sakai, Yusaku; Ishino, Haruka; Hyakumachi, Mitsuro; Yamamoto, Yoshiharu Y


    Hydrogen peroxide acts as a signaling molecule mediating the acquisition of tolerance to both biotic and abiotic stresses. Identification of marker genes for H2O2 response could help to intercept the signaling network of stress response of plants. Here, we describe application of marker genes for H2O2 responses to monitoring several abiotic stress responses. Arabidopsis plants were treated with UV-B, high light, and cold stresses, where involvement of H2O2-mediated signaling is known or suggested. Monitoring of these stress responses with molecular markers using quantitative real-time RT-PCR can detect landmark events in the sequential stress responses. These methods can be used for analysis of mutants and transgenic plants to examine natural H2O2 responses that are involved in environmental adaptation.

  18. A comprehensive resource of drought- and salinity- responsive ESTs for gene discovery and marker development in chickpea (Cicer arietinum L.).

    Varshney, Rajeev K; Hiremath, Pavana J; Lekha, Pazhamala; Kashiwagi, Junichi; Balaji, Jayashree; Deokar, Amit A; Vadez, Vincent; Xiao, Yongli; Srinivasan, Ramamurthy; Gaur, Pooran M; Siddique, Kadambot Hm; Town, Christopher D; Hoisington, David A


    Chickpea (Cicer arietinum L.), an important grain legume crop of the world is seriously challenged by terminal drought and salinity stresses. However, very limited number of molecular markers and candidate genes are available for undertaking molecular breeding in chickpea to tackle these stresses. This study reports generation and analysis of comprehensive resource of drought- and salinity-responsive expressed sequence tags (ESTs) and gene-based markers. A total of 20,162 (18,435 high quality) drought- and salinity- responsive ESTs were generated from ten different root tissue cDNA libraries of chickpea. Sequence editing, clustering and assembly analysis resulted in 6,404 unigenes (1,590 contigs and 4,814 singletons). Functional annotation of unigenes based on BLASTX analysis showed that 46.3% (2,965) had significant similarity (chickpea. Of 2,965 (46.3%) significant unigenes, only 2,071 (32.3%) unigenes could be functionally categorised according to Gene Ontology (GO) descriptions. A total of 2,029 sequences containing 3,728 simple sequence repeats (SSRs) were identified and 177 new EST-SSR markers were developed. Experimental validation of a set of 77 SSR markers on 24 genotypes revealed 230 alleles with an average of 4.6 alleles per marker and average polymorphism information content (PIC) value of 0.43. Besides SSR markers, 21,405 high confidence single nucleotide polymorphisms (SNPs) in 742 contigs (with > or = 5 ESTs) were also identified. Recognition sites for restriction enzymes were identified for 7,884 SNPs in 240 contigs. Hierarchical clustering of 105 selected contigs provided clues about stress- responsive candidate genes and their expression profile showed predominance in specific stress-challenged libraries. Generated set of chickpea ESTs serves as a resource of high quality transcripts for gene discovery and development of functional markers associated with abiotic stress tolerance that will be helpful to facilitate chickpea breeding. Mapping of

  19. Ehrlichia chaffeensis TRP32 is a Nucleomodulin that Directly Regulates Expression of Host Genes Governing Differentiation and Proliferation.

    Farris, Tierra R; Dunphy, Paige S; Zhu, Bing; Kibler, Clayton E; McBride, Jere W


    Ehrlichia chaffeensis is an obligately intracellular bacterium that reprograms the mononuclear phagocyte through diverse effector-host interactions to modulate numerous host cell processes, including transcription. In a previous study, we reported that E. chaffeensis TRP32, a type 1 secreted effector, interacts with multiple host nucleus-associated proteins and also auto-activates reporter gene expression in yeast. In this study, we demonstrate that TRP32 is a nucleomodulin that binds host DNA and alters host gene transcription. TRP32 enters the host cell nucleus via a noncanonical translocation mechanism that involves phosphorylation of Y179 located in a C-terminal tri-tyrosine motif. Both genistein and mutation of Y179 inhibited TRP32 nuclear entry. An electromobility shift assay (EMSA) demonstrated TRP32 host DNA binding via its tandem repeat domain. TRP32 DNA binding and motif preference were further confirmed by supershift assays, as well as competition and mutant probe analyses. Using ChIP-Seq, we determined that TRP32 binds a G-rich motif primarily within ±500 bp of the gene transcription start site. An ontology analysis identified genes involved in processes such as immune cell differentiation, chromatin remodeling, and RNA transcription and processing, as primary TRP32 targets. TRP32 bound genes (n=1223) were distributed on all chromosomes and included several global regulators of proliferation and inflammation such as FOS and JUN, AKT3 and NRAS, and non-coding RNA genes, miRNA 21 and miRNA 142. TRP32 target genes were differentially regulated during infection, the majority of which were repressed, and direct repression/activation of these genes by TRP32 was confirmed in vitro with a cellular luciferase reporter assay.

  20. Defining the expression of marker genes in equine mesenchymal stromal cells

    Deborah J Guest


    Full Text Available Deborah J Guest1, Jennifer C Ousey1, Matthew RW Smith21Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, CB8 7UU; 2Reynolds House Referrals, Greenwood Ellis and Partners, 166 High Street, Newmarket, Suffolk, CB8 9WS, UKAbstract: Mesenchymal stromal (MS cells have been derived from multiple sources in the horse including bone marrow, adipose tissue and umbilical cord blood. To date these cells have been investigated for their differentiation potential and are currently being used to treat damage to horse musculoskeletal tissues. However, no work has been done in horse MS cells to examine the expression profile of proteins and cell surface antigens that are expressed in human MS cells. The identification of such profiles in the horse will allow the comparison of putative MS cells isolated from different laboratories and different tissues. At present it is difficult to ascertain whether equivalent cells are being used in different reports. Here, we report on the expression of a range of markers used to define human MS cells. Using immunocytochemistry we show that horse MS cells homogenously express collagens, alkaline phosphatase activity, CD44, CD90 and CD29. In contrast, CD14, CD79α and the embryonic stem cell markers Oct-4, SSEA (stage specific embryonic antigen -1, -3, -4, TRA (tumor rejection antigen -1–60 and -1–81 are not expressed. The MS cells also express MHC class I antigens but do not express class II antigens, although they are inducible by treatment with interferon gamma (IFN-γ.Keywords: mesenchymal stem cells, equine, gene expression

  1. Copy number gain of VCX, X-linked multi-copy gene, leads to cell proliferation and apoptosis during spermatogenesis

    Ji, Juan; Qin, Yufeng; Wang, Rong; Huang, Zhenyao; Zhang, Yan; Zhou, Ran; Song, Ling; Ling, Xiufeng; Hu, Zhibin; Miao, Dengshun; Shen, Hongbing; Xia, Yankai; Wang, Xinru; Lu, Chuncheng


    Male factor infertility affects one-sixth of couples worldwide, and non-obstructive azoospermia (NOA) is one of the most severe forms. In recent years there has been increasing evidence to implicate the participation of X chromosome in the process of spermatogenesis. To uncover the roles of X-linked multi-copy genes in spermatogenesis, we performed systematic analysis of X-linked gene copy number variations (CNVs) and Y chromosome haplogrouping in 447 idiopathic NOA patients and 485 healthy controls. Interestingly, the frequency of individuals with abnormal level copy of Variable charge, X-linked (VCX) was significantly different between cases and controls after multiple test correction (p = 5.10 × 10−5). To discriminate the effect of gain/loss copies in these genes, we analyzed the frequency of X-linked multi-copy genes in subjects among subdivided groups. Our results demonstrated that individuals with increased copy numbers of Nuclear RNA export factor 2 (NXF2) (p = 9.21 × 10−8) and VCX (p = 1.97 × 10−4) conferred the risk of NOA. In vitro analysis demonstrated that increasing copy number of VCX could upregulate the gene expression and regulate cell proliferation and apoptosis. Our study establishes a robust association between the VCX CNVs and NOA risk. PMID:27705943

  2. Copy number gain of VCX, X-linked multi-copy gene, leads to cell proliferation and apoptosis during spermatogenesis.

    Ji, Juan; Qin, Yufeng; Wang, Rong; Huang, Zhenyao; Zhang, Yan; Zhou, Ran; Song, Ling; Ling, Xiufeng; Hu, Zhibin; Miao, Dengshun; Shen, Hongbing; Xia, Yankai; Wang, Xinru; Lu, Chuncheng


    Male factor infertility affects one-sixth of couples worldwide, and non-obstructive azoospermia (NOA) is one of the most severe forms. In recent years there has been increasing evidence to implicate the participation of X chromosome in the process of spermatogenesis. To uncover the roles of X-linked multi-copy genes in spermatogenesis, we performed systematic analysis of X-linked gene copy number variations (CNVs) and Y chromosome haplogrouping in 447 idiopathic NOA patients and 485 healthy controls. Interestingly, the frequency of individuals with abnormal level copy of Variable charge, X-linked (VCX) was significantly different between cases and controls after multiple test correction (p = 5.10 × 10-5). To discriminate the effect of gain/loss copies in these genes, we analyzed the frequency of X-linked multi-copy genes in subjects among subdivided groups. Our results demonstrated that individuals with increased copy numbers of Nuclear RNA export factor 2 (NXF2) (p = 9.21 × 10-8) and VCX (p = 1.97 × 10-4) conferred the risk of NOA. In vitro analysis demonstrated that increasing copy number of VCX could upregulate the gene expression and regulate cell proliferation and apoptosis. Our study establishes a robust association between the VCX CNVs and NOA risk.

  3. Peroxisome proliferator-activated receptor subtype- and cell-type-specific activation of genomic target genes upon adenoviral transgene delivery

    Nielsen, Ronni; Grøntved, Lars; Stunnenberg, Hendrik G


    Investigations of the molecular events involved in activation of genomic target genes by peroxisome proliferator-activated receptors (PPARs) have been hampered by the inability to establish a clean on/off state of the receptor in living cells. Here we show that the combination of adenoviral...... delivery and chromatin immunoprecipitation (ChIP) is ideal for dissecting these mechanisms. Adenoviral delivery of PPARs leads to a rapid and synchronous expression of the PPAR subtypes, establishment of transcriptional active complexes at genomic loci, and immediate activation of even silent target genes....... We demonstrate that PPARgamma2 possesses considerable ligand-dependent as well as independent transactivation potential and that agonists increase the occupancy of PPARgamma2/retinoid X receptor at PPAR response elements. Intriguingly, by direct comparison of the PPARs (alpha, gamma, and beta...

  4. Exploring the Transcriptome Landscape of Pomegranate Fruit Peel for Natural Product Biosynthetic Gene and SSR Marker Discovery(F).

    Ono, Nadia Nicole; Britton, Monica Therese; Fass, Joseph Nathaniel; Nicolet, Charles Meyer; Lin, Dawei; Tian, Li


    Pomegranate fruit peel is rich in bioactive plant natural products, such as hydrolyzable tannins and anthocyanins. Despite their documented roles in human nutrition and fruit quality, genes involved in natural product biosynthesis have not been cloned from pomegranate and very little sequence information is available on pomegranate in the public domain. Shotgun transcriptome sequencing of pomegranate fruit peel cDNA was performed using RNA-Seq on the Illumina Genome Analyzer platform. Over 100 million raw sequence reads were obtained and assembled into 9,839 transcriptome assemblies (TAs) (>200 bp). Candidate genes for hydrolyzable tannin, anthocyanin, flavonoid, terpenoid and fatty acid biosynthesis and/or regulation were identified. Three lipid transfer proteins were obtained that may contribute to the previously reported IgE reactivity of pomegranate fruit extracts. In addition, 115 SSR markers were identified from the pomegranate fruit peel transcriptome and primers were designed for 77 SSR markers. The pomegranate fruit peel transcriptome set provides a valuable platform for natural product biosynthetic gene and SSR marker discovery in pomegranate. This work also demonstrates that next-generation transcriptome sequencing is an economical and effective approach for investigating natural product biosynthesis, identifying genes controlling important agronomic traits, and discovering molecular markers in non-model specialty crop species.

  5. Tissue-specifically regulated site-specific excision of selectable marker genes in bivalent insecticidal, genetically-modified rice.

    Hu, Zhan; Ding, Xuezhi; Hu, Shengbiao; Sun, Yunjun; Xia, Liqiu


    Marker-free, genetically-modified rice was created by the tissue-specifically regulated Cre/loxP system, in which the Cre recombinase gene and hygromycin phosphotransferase gene (hpt) were flanked by two directly oriented loxP sites. Cre expression was activated by the tissue-specific promoter OsMADS45 in flower or napin in seed, resulting in simultaneous excision of the recombinase and marker genes. Segregation of T1 progeny was performed to select recombined plants. The excision was confirmed by PCR, Southern blot and sequence analyses indicating that efficiency varied from 10 to 53 % for OsMADS45 and from 12 to 36 % for napin. The expression of cry1Ac and vip3A was detected by RT-PCR analysis in marker-free transgenic rice. These results suggested that our tissue-specifically regulated Cre/loxP system could auto-excise marker genes from transgenic rice and alleviate public concerns about the security of GM crops.

  6. AFLP markers for the R-gene in the flea beetle, Phyllotreta nemorum, conferring resistance to defenses in Barbarea vulgaris

    Breuker, C.J.; Victoir, K.; Jong, de P.W.; Meijden, van der E.; Brakefield, P.M.; Vrieling, K.


    A so-called R-gene renders the yellow-striped flea beetle Phyllotreta nemorum L. (Coleoptera: Chrysomelidae: Alticinae) resistant to the defenses of the yellow rocket Barbarea vulgaris R.Br. (Brassicacea) and enables it to use it as a host plant in Denmark. In this study, genetic markers for an auto

  7. Linkage study of nonsyndromic cleft lip with or without cleft palate using candidate genes and mapped polymorphic markers

    Stein, J.D.; Nelson, L.D.; Conner, B.J. [Univ. of Texas, Houston (United States)] [and others


    Nonsyndromic cleft lip with or without cleft palate (CL(P)) involves fusion or growth failure of facial primordia during development. Complex segregation analysis of clefting populations suggest that an autosomal dominant gene may play a role in this common craniofacial disorder. We have ascertained 16 multigenerational families with CL(P) and tested linkage to 29 candidate genes and 139 mapped short tandem repeat markers. The candidate genes were selected based on their expression in craniofacial development or were identified through murine models. These include: TGF{alpha}, TGF{beta}1, TGF{beta}2, TGF{beta}3, EGF, EGFR, GRAS, cMyc, FGFR, Jun, JunB, PDFG{alpha}, PDGF{beta}, IGF2R, GCR Hox7, Hox8, Hox2B, twirler, 5 collagen and 3 extracellular matrix genes. Linkage was tested assuming an autosomal dominant model with sex-specific decreased penetrance. Linkage to all of the candidate loci was excluded in 11 families. RARA was tested and was not informative. However, haplotype analysis of markers flanking RARA on 17q allowed exclusion of this candidate locus. We have previously excluded linkage to 61 STR markers in 11 families. Seventy-eight mapped short tandem repeat markers have recently been tested in 16 families and 30 have been excluded. The remaining are being analyzed and an exclusion map is being developed based on the entire study results.

  8. Exploring the Transcriptome Landscape of Pomegranate Fruit Peel for Natural Product Biosynthetic Gene and SSR Marker Discovery

    Nadia Nicole Ono; Monica Therese Britton; Joseph Nathaniel Fass; Charles Meyer Nicolet; Dawei Lin; Li Tian


    Pomegranate fruit peel is rich in bioactive plant natural products,such as hydrolyzable tannins and anthocyanins.Despite their documented roles in human nutrition and fruit quality,genes involved in natural product biosynthesis have not been cloned from pomegranate and very little sequence information is available on pomegranate in the public domain.Shotgun transcriptome sequencing of pomegranate fruit peel cDNA was performed using RNA-Seq on the Illumina Genome Analyzer platform.Over 100 million raw sequence reads were obtained and assembled into 9,839 transcriptome assemblies (TAs) (>200 bp).Candidate genes for hydrolyzable tannin,anthocyanin,flavonoid,terpenoid and fatty acid biosynthesis and/or regulation were identified.Three lipid transfer proteins were obtained that may contribute to the previously reported IgE reactivity of pomegranate fruit extracts.In addition,115 SSR markers were identified from the pomegranate fruit peel transcriptome and primers were designed for 77 SSR markers.The pomegranate fruit peel transcriptome set provides a valuable platform for natural product biosynthetic gene and SSR marker discovery in pomegranate.This work also demonstrates that next-generation transcriptome sequencing is an economical and effective approach for investigating natural product biosynthesis,identifying genes controlling important agronomic traits,and discovering molecular markers in non-model specialty crop species.

  9. Neural differentiation potential of human bone marrow-derived mesenchymal stromal cells: misleading marker gene expression

    Montzka Katrin


    Full Text Available Abstract Background In contrast to pluripotent embryonic stem cells, adult stem cells have been considered to be multipotent, being somewhat more restricted in their differentiation capacity and only giving rise to cell types related to their tissue of origin. Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries. Such reports have been based on the detection of neural-related proteins by the differentiated MSCs. In order to assess the potential of human adult MSCs to undergo true differentiation to a neural lineage and to determine the degree of homogeneity between donor samples, we have used RT-PCR and immunocytochemistry to investigate the basal expression of a range of neural related mRNAs and proteins in populations of non-differentiated MSCs obtained from 4 donors. Results The expression analysis revealed that several of the commonly used marker genes from other studies like nestin, Enolase2 and microtubule associated protein 1b (MAP1b are already expressed by undifferentiated human MSCs. Furthermore, mRNA for some of the neural-related transcription factors, e.g. Engrailed-1 and Nurr1 were also strongly expressed. However, several other neural-related mRNAs (e.g. DRD2, enolase2, NFL and MBP could be identified, but not in all donor samples. Similarly, synaptic vesicle-related mRNA, STX1A could only be detected in 2 of the 4 undifferentiated donor hMSC samples. More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression. Conclusion The present study highlights the existence of an inter-donor variability of expression of neural-related markers in human MSC samples that has not previously been described. This donor-related heterogeneity might influence the reproducibility of transdifferentiation protocols as

  10. The effect of missing marker genotypes on the accuracy of gene-assisted breeding value estimation: a comparison of methods.

    Mulder, H A; Meuwissen, T H E; Calus, M P L; Veerkamp, R F


    In livestock populations, missing genotypes on a large proportion of the animals is a major problem when implementing gene-assisted breeding value estimation for genes with known effect. The objective of this study was to compare different methods to deal with missing genotypes on accuracy of gene-assisted breeding value estimation for identified bi-allelic genes using Monte Carlo simulation. A nested full-sib half-sib structure was simulated with a mixed inheritance model with one bi-allelic quantitative trait loci (QTL) and a polygenic effect due to infinite number of polygenes. The effect of the QTL was included in gene-assisted BLUP either by random regression on predicted gene content, i.e. the number of positive alleles, or including haplotype effects in the model with an inverse IBD matrix to account for identity-by-descent relationships between haplotypes using linkage analysis information (IBD-LA). The inverse IBD matrix was constructed using segregation indicator probabilities obtained from multiple marker iterative peeling. Gene contents for unknown genotypes were predicted using either multiple marker iterative peeling or mixed model methodology. For both methods, gene-assisted breeding value estimation increased accuracies of total estimated breeding value (EBV) with 0% to 22% for genotyped animals in comparison to conventional breeding value estimation. For animals that were not genotyped, the increase in accuracy was much lower (0% to 5%), but still substantial when the heritability was 0.1 and when the QTL explained at least 15% of the genetic variance. Regression on predicted gene content yielded higher accuracies than IBD-LA. Allele substitution effects were, however, overestimated, especially when only sires and males in the last generation were genotyped. For juveniles without phenotypic records and traits measured only on females, the superiority of regression on gene content over IBD-LA was larger than when all animals had phenotypes. Missing

  11. Effects of recombinant adenoviral vector containing IRE1α gene on proliferation and apoptosis of ATDC5 stem cells

    Xiang-zhu LI


    Full Text Available Objective To construct the recombinant adenoviral vector containing human IRE1α (type I transmembrane protein kinase/endoribonucleasegene, and investigate its effects on proliferation and apoptosis of ATDC5 stem cells. Methods  By using pAdEasyTM adenovirus vector system, the recombinant shuttle vectors of IRE1α full-length gene(pAdTrack-IRE1αand RNase+Kinasedomain(pAdTrack-R+Kwere constructed, and then transferred with pAdEasy-1 to generate recombinant adenovirus plasmid pAd-IRE1α and pAd-R+K by electroporation method. Subsequently, the plasmids were transfected into HEK-293 cells to pack and amplify the recombinant adenovirus Ad-IRE1α and Ad-R+K. The expression of recombinant adenovirus was detected by PCR. The ATDC5 cells wereinfected in vitro by recombinant adenovirus Ad-IRE1α and Ad-R+K, the infection efficiency of green fluorescent protein(GFPwas observed, and the influence of Ad-IRE1α and Ad-R+K on the proliferation and apoptosis of ATDC5 cells under endoplasmic reticulum stress(ERS or non-ERS was detected by flow cytometry(FCM. Results Restriction endonuclease digestion analysis and PCR indicated that the recombinant adenovirus vector Ad-IRE1α andAd-R+K was successfully constructed. FCM detection showed that under ERS conditions, the G1 phasedcreased and S phase increased in ATDC5 cells after transfected by Ad-IRE1α and Ad-R+K, meanwhile the apoptosis rate increased significantly(P<0.05. Conclusion Infection of recombinant adenovirus containing IRE1α gene may promote the proliferation and apoptosis of ATDC5cells.

  12. Antioxidative Dietary Compounds Modulate Gene Expression Associated with Apoptosis, DNA Repair, Inhibition of Cell Proliferation and Migration

    Likui Wang


    Full Text Available Many dietary compounds are known to have health benefits owing to their antioxidative and anti-inflammatory properties. To determine the molecular mechanism of these food-derived compounds, we analyzed their effect on various genes related to cell apoptosis, DNA damage and repair, oxidation and inflammation using in vitro cell culture assays. This review further tests the hypothesis proposed previously that downstream products of COX-2 (cyclooxygenase-2 called electrophilic oxo-derivatives induce antioxidant responsive elements (ARE, which leads to cell proliferation under antioxidative conditions. Our findings support this hypothesis and show that cell proliferation was inhibited when COX-2 was down-regulated by polyphenols and polysaccharides. Flattened macrophage morphology was also observed following the induction of cytokine production by polysaccharides extracted from viili, a traditional Nordic fermented dairy product. Coix lacryma-jobi (coix polysaccharides were found to reduce mitochondrial membrane potential and induce caspase-3- and 9-mediated apoptosis. In contrast, polyphenols from blueberries were involved in the ultraviolet-activated p53/Gadd45/MDM2 DNA repair system by restoring the cell membrane potential. Inhibition of hypoxia-inducible factor-1 by saponin extracts of ginsenoside (Ginsen and Gynostemma and inhibition of S100A4 by coix polysaccharides inhibited cancer cell migration and invasion. These observations suggest that antioxidants and changes in cell membrane potential are the major driving forces that transfer signals through the cell membrane into the cytosol and nucleus, triggering gene expression, changes in cell proliferation and the induction of apoptosis or DNA repair.

  13. Docosahexaenoyl ethanolamide improves glucose uptake and alters endocannabinoid system gene expression in proliferating and differentiating C2C12 myoblasts

    Jeffrey eKim


    Full Text Available Skeletal muscle is a major storage site for glycogen and a focus for understanding insulin resistance and type-2-diabetes. New evidence indicates that overactivation of the peripheral endocannabinoid system (ECS in skeletal muscle diminishes insulin sensitivity. Specific n-6 and n-3 polyunsaturated fatty acids (PUFA are precursors for the biosynthesis of ligands that bind to and activate the cannabinoid receptors. The function of the ECS and action of PUFA in skeletal muscle glucose uptake was investigated in proliferating and differentiated C2C12 myoblasts treated with either 25µM of arachidonate (AA or docosahexaenoate (DHA, 25µM of EC [anandamide (AEA, 2-arachidonoylglycerol (2-AG, docosahexaenoylethanolamide (DHEA], 1µM of CB1 antagonist NESS0327, and CB2 antagonist AM630. Compared to the BSA vehicle control cell cultures in both proliferating and differentiated myoblasts those treated with DHEA, the EC derived from the n-3 PUFA DHA, had higher 24 h glucose uptake, while AEA and 2-AG, the EC derived from the n-6 PUFA AA, had lower basal glucose uptake. Adenylyl cyclase mRNA was higher in myoblasts treated with DHA in both proliferating and differentiated states while those treated with AEA or 2-AG were lower compared to the control cell cultures. Western blot and qPCR analysis showed higher expression of the cannabinoid receptors in differentiated myoblasts treated with DHA while the opposite was observed with AA. These findings indicate a compensatory effect of DHA and DHEA compared to AA-derived ligands on the ECS and associated ECS gene expression and higher glucose uptake in myoblasts.Key Words: endocannabinoid system •C2C12 myoblasts cannabinoid receptors glucose uptake gene expression DHEA • polyunsaturated fatty acids

  14. Multi-class clustering of cancer subtypes through SVM based ensemble of pareto-optimal solutions for gene marker identification.

    Anirban Mukhopadhyay

    Full Text Available With the advancement of microarray technology, it is now possible to study the expression profiles of thousands of genes across different experimental conditions or tissue samples simultaneously. Microarray cancer datasets, organized as samples versus genes fashion, are being used for classification of tissue samples into benign and malignant or their subtypes. They are also useful for identifying potential gene markers for each cancer subtype, which helps in successful diagnosis of particular cancer types. In this article, we have presented an unsupervised cancer classification technique based on multiobjective genetic clustering of the tissue samples. In this regard, a real-coded encoding of the cluster centers is used and cluster compactness and separation are simultaneously optimized. The resultant set of near-Pareto-optimal solutions contains a number of non-dominated solutions. A novel approach to combine the clustering information possessed by the non-dominated solutions through Support Vector Machine (SVM classifier has been proposed. Final clustering is obtained by consensus among the clusterings yielded by different kernel functions. The performance of the proposed multiobjective clustering method has been compared with that of several other microarray clustering algorithms for three publicly available benchmark cancer datasets. Moreover, statistical significance tests have been conducted to establish the statistical superiority of the proposed clustering method. Furthermore, relevant gene markers have been identified using the clustering result produced by the proposed clustering method and demonstrated visually. Biological relationships among the gene markers are also studied based on gene ontology. The results obtained are found to be promising and can possibly have important impact in the area of unsupervised cancer classification as well as gene marker identification for multiple cancer subtypes.

  15. Gene targeting in the red alga Cyanidioschyzon merolae: single- and multi-copy insertion using authentic and chimeric selection markers.

    Fujiwara, Takayuki; Ohnuma, Mio; Yoshida, Masaki; Kuroiwa, Tsuneyoshi; Hirano, Tatsuya


    The unicellular red alga Cyanidioschyzon merolae is an emerging model organism for studying organelle division and inheritance: the cell is composed of an extremely simple set of organelles (one nucleus, one mitochondrion and one chloroplast), and their genomes are completely sequenced. Although a fruitful set of cytological and biochemical methods have now been developed, gene targeting techniques remain to be fully established in this organism. Thus far, only a single selection marker, URA Cm-Gs , has been available that complements the uracil-auxotrophic mutant M4. URA Cm-Gs , a chimeric URA5.3 gene of C. merolae and the related alga Galdieria sulphuraria, was originally designed to avoid gene conversion of the mutated URA5.3 allele in the parental strain M4. Although an early example of targeted gene disruption by homologous recombination was reported using this marker, the genome structure of the resultant transformants had never been fully characterized. In the current study, we showed that the use of the chimeric URA Cm-Gs selection marker caused multicopy insertion at high frequencies, accompanied by undesired recombination events at the targeted loci. The copy number of the inserted fragments was variable among the transformants, resulting in high yet uneven levels of transgene expression. In striking contrast, when the authentic URA5.3 gene (URA Cm-Cm ) was used as a selection marker, efficient single-copy insertion was observed at the targeted locus. Thus, we have successfully established a highly reliable and reproducible method for gene targeting in C. merolae. Our method will be applicable to a number of genetic manipulations in this organism, including targeted gene disruption, replacement and tagging.

  16. Transferring Translucent Endosperm Mutant Gene Wx-mq and Rice Stripe Disease Resistance Gene Stv-bi by Marker-Assisted Selection in Rice (Oryza sativa)

    YAO Shu; CHEN Tao; ZHANG Ya-dong; ZHU Zhen; ZHAO Ling; ZHAO Qing-yong; ZHOU Li-hui; WANG Cai-lin


    A high-yielding japonica rice variety,Wuyunjing 7,bred in Jiangsu Province,China as a female parent was crossed with a Japanese rice variety Kantou 194,which carries a rice stripe disease resistance gene Stv-b1 and a translucent endosperm mutant gene Wx-mq.From F2 generations,a sequence characterized amplified region (SCAR) marker tightly linked with Stv-b1 and a cleaved amplified polymorphic sequence (CAPS) marker for Wx-mq were used for marker-assisted selection.Finally,a new japonica rice line,Ning 9108,with excellent agronomic traits was obtained by multi-generational selection on stripe disease resistance and endosperm appearance.The utilization of the markers from genes related to rice quality and disease resistance was helpful not only for establishing a marker-assisted selection system of high-quality and disease resistance for rice but also for providing important intermediate materials and rapid selection method for good quality,disease resistance and high yield in rice breeding.

  17. Fine Mapping of a Clubroot Resistance Gene in Chinese Cabbage Using SNP Markers Identified from Bulked Segregant RNA Sequencing

    Huang, Zhen; Peng, Gary; Liu, Xunjia; Deora, Abhinandan; Falk, Kevin C.; Gossen, Bruce D.; McDonald, Mary R.; Yu, Fengqun


    Clubroot, caused by Plasmodiophora brassicae, is an important disease of canola (Brassica napus) in western Canada and worldwide. In this study, a clubroot resistance gene (Rcr2) was identified and fine mapped in Chinese cabbage cv. “Jazz” using single-nucleotide polymorphisms (SNP) markers identified from bulked segregant RNA sequencing (BSR-Seq) and molecular markers were developed for use in marker assisted selection. In total, 203.9 million raw reads were generated from one pooled resistant (R) and one pooled susceptible (S) sample, and >173,000 polymorphic SNP sites were identified between the R and S samples. One significant peak was observed between 22 and 26 Mb of chromosome A03, which had been predicted by BSR-Seq to contain the causal gene Rcr2. There were 490 polymorphic SNP sites identified in the region. A segregating population consisting of 675 plants was analyzed with 15 SNP sites in the region using the Kompetitive Allele Specific PCR method, and Rcr2 was fine mapped between two SNP markers, SNP_A03_32 and SNP_A03_67 with 0.1 and 0.3 cM from Rcr2, respectively. Five SNP markers co-segregated with Rcr2 in this region. Variants were identified in 14 of 36 genes annotated in the Rcr2 target region. The numbers of poly variants differed among the genes. Four genes encode TIR-NBS-LRR proteins and two of them Bra019410 and Bra019413, had high numbers of polymorphic variants and so are the most likely candidates of Rcr2. PMID:28894454

  18. Identification of Random Amplified Polymorphic DNA Markers Linked to the Co-4 Resistance Gene to Colletotrichum lindemuthianum in Common Bean.

    de Arruda, M C; Alzate-Marin, A L; Chagas, J M; Moreira, M A; de Barros, E G


    ABSTRACT New cultivars of the common bean (Phaseolus vulgaris) with durable resistance to anthracnose can be developed by pyramiding major resistance genes using marker-assisted selection. To this end, it is necessary to identify sources of resistance and molecular markers tightly linked to the resistance genes. The objectives of this work were to study the inheritance of resistance to anthracnose in the cultivar TO (carrying the Co-4 gene), to identify random amplified polymorphic DNA (RAPD) markers linked to Co-4, and to introgress this gene in the cultivar Rudá. Populations F(1), F(2), F(2:3), BC(1)s, and BC(1)r from the cross Rudá x TO were inoculated with race 65 of Colletotrichum lindemuthianum, causal agent of bean anthracnose. The phenotypic ratios (resistant/susceptible) were 3:1 in the F(2) population, 1:1 in the BC(1)s, and 1:0 in the BC(1)r, confirming that resistance to anthracnose in the cultivar TO was monogenic and dominant. Six RAPD markers linked to the Co-4 gene were identified, four in the coupling phase: OPY20(830C) (0.0 centimorgan [cM]), OPC08(900C) (9.7 cM), OPI16(850C) (14.3 cM), and OPJ01(1,380C) (18.1 cM); and two in the repulsion phase: OPB03(1,800T) (3.7 cM) and OPA18(830T) (17.4 cM). OPY20(830C) and OPB03(1,800T), used in association as a codominant pair, allowed the identification of the three genotypic classes with a high degree of confidence. Marker OPY20(830C), which is tightly linked to Co-4, is being used to assist in breeding for resistance to anthracnose.

  19. GAP1, a novel selection and counter-selection marker for multiple gene disruptions in Saccharomyces cerevisiae

    Regenberg, Birgitte; Hansen, J.


    We report on the use of a new homologous marker for use in multiple gene deletions in S, cerevisiae, the general amino acid permease gene (GAP1), A GAP1 strain can utilize L-citrulline as the sole nitrogen source but cannot grow in the presence of the toxic amino acid D-histidine, L-citrulline as......We report on the use of a new homologous marker for use in multiple gene deletions in S, cerevisiae, the general amino acid permease gene (GAP1), A GAP1 strain can utilize L-citrulline as the sole nitrogen source but cannot grow in the presence of the toxic amino acid D-histidine, L......-citrulline as well as D-histidine uptake is mediated solely by the general amino acid permease, and a gap1 strain is therefore able to grow in the presence of D-histidine but cannot utilize L-citrulline, Gene disruption is effected by transforming a gap1 strain with a gene cassette generated by PCR, containing GAP1...... the GAP1 gene. This is caused by recombination between two Salmonella typuimurium hisG direct repeats embracing GAP1, and will result in a sub-population of gap1 cells. Such cells are selected on a medium containing D-histidine, and may subsequently be used for a second gene disruption. Hence, multiple...

  20. The peroxisome proliferator-activated receptor N-terminal domain controls isotype-selective gene expression and adipogenesis.

    Hummasti, Sarah; Tontonoz, Peter


    Peroxisome proliferator-activated receptors (PPARgamma, PPARalpha, and PPARdelta) are important regulators of lipid metabolism. Although they share significant structural similarity, the biological effects associated with each PPAR isotype are distinct. For example, PPARalpha and PPARdelta regulate fatty acid catabolism, whereas PPARgamma controls lipid storage and adipogenesis. The different functions of PPARs in vivo can be explained at least in part by the different tissue distributions of the three receptors. The question of whether the receptors have different intrinsic activities and regulate distinct target genes, however, has not been adequately explored. We have engineered cell lines that express comparable amounts of each receptor. Transcriptional profiling of these cells in the presence of selective agonists reveals partially overlapping but distinct patterns of gene regulation by the three PPARs. Moreover, analysis of chimeric receptors points to the N terminus of each receptor as the key determinant of isotype-selective gene expression. For example, the N terminus of PPARgamma confers the ability to promote adipocyte differentiation when fused to the PPARdelta DNA binding domain and ligand binding domain, whereas the N terminus of PPARdelta leads to the inappropriate expression of fatty acid oxidation genes in differentiated adipocytes when fused to PPARgamma. Finally, we demonstrate that the N terminus of each receptor functions in part to limit receptor activity because deletion of the N terminus leads to nonselective activation of target genes. A more detailed understanding of the mechanisms by which the individual PPARs differentially regulate gene expression should aid in the design of more effective drugs, including tissue- and target gene-selective PPAR modulators.

  1. A Cross-Species Gene Expression Marker-Based Genetic Map and QTL Analysis in Bambara Groundnut

    Hui Hui Chai


    Full Text Available Bambara groundnut (Vigna subterranea (L. Verdc. is an underutilised legume crop, which has long been recognised as a protein-rich and drought-tolerant crop, used extensively in Sub-Saharan Africa. The aim of the study was to identify quantitative trait loci (QTL involved in agronomic and drought-related traits using an expression marker-based genetic map based on major crop resources developed in soybean. The gene expression markers (GEMs were generated at the (unmasked probe-pair level after cross-hybridisation of bambara groundnut leaf RNA to the Affymetrix Soybean Genome GeneChip. A total of 753 markers grouped at an LOD (Logarithm of odds of three, with 527 markers mapped into linkage groups. From this initial map, a spaced expression marker-based genetic map consisting of 13 linkage groups containing 218 GEMs, spanning 982.7 cM (centimorgan of the bambara groundnut genome, was developed. Of the QTL detected, 46% were detected in both control and drought treatment populations, suggesting that they are the result of intrinsic trait differences between the parental lines used to construct the cross, with 31% detected in only one of the conditions. The present GEM map in bambara groundnut provides one technically feasible route for the translation of information and resources from major and model plant species to underutilised and resource-poor crops.

  2. Sex determination in 58 bird species and evaluation of CHD gene as a universal molecular marker in bird sexing.

    Vucicevic, Milos; Stevanov-Pavlovic, Marija; Stevanovic, Jevrosima; Bosnjak, Jasna; Gajic, Bojan; Aleksic, Nevenka; Stanimirovic, Zoran


    The aim of this research was to test the CHD gene (Chromo Helicase DNA-binding gene) as a universal molecular marker for sexing birds of relatively distant species. The CHD gene corresponds to the aim because of its high degree of conservation and different lengths in Z and W chromosomes due to different intron sizes. DNA was isolated from feathers and the amplification of the CHD gene was performed with the following sets of polymerase chain reaction (PCR) primers: 2550F/2718R and P2/P8. Sex determination was attempted in 284 samples of 58 bird species. It was successful in 50 bird species; in 16 of those (Alopochen aegyptiacus, Ara severus, Aratinga acuticaudata, Bucorvus leadbeateri, Cereopsis novaehollandiae, Columba arquatrix, Corvus corax, C. frugilegus, Cyanoliseus patagonus, Guttera plumifera, Lamprotornis superbus, Milvus milvus, Neophron percnopterus, Ocyphaps lophotes, Podiceps cristatus, and Poicephalus senegalus), it was carried out for the first time using molecular markers and PCR. It is reasonable to assume that extensive research is necessary to define the CHD gene as a universal molecular marker for successful sex determination in all bird species (with exception of ratites). The results of this study may largely contribute to the aim.

  3. CD177: A member of the Ly-6 gene superfamily involved with neutrophil proliferation and polycythemia vera

    Bettinotti Maria


    Full Text Available Abstract Genes in the Leukocyte Antigen 6 (Ly-6 superfamily encode glycosyl-phosphatidylinositol (GPI anchored glycoproteins (gp with conserved domains of 70 to 100 amino acids and 8 to 10 cysteine residues. Murine Ly-6 genes encode important lymphocyte and hematopoietic stem cell antigens. Recently, a new member of the human Ly-6 gene superfamily has been described, CD177. CD177 is polymorphic and has at least two alleles, PRV-1 and NB1. CD177 was first described as PRV-1, a gene that is overexpressed in neutrophils from approximately 95% of patients with polycythemia vera and from about half of patients with essential thrombocythemia. CD177 encodes NB1 gp, a 58–64 kD GPI gp that is expressed by neutrophils and neutrophil precursors. NB1 gp carries Human Neutrophil Antigen (HNA-2a. Investigators working to identify the gene encoding NB1 gp called the CD177 allele they described NB1. NB1 gp is unusual in that neutrophils from some healthy people lack the NB1 gp completely and in most people NB1 gp is expressed by a subpopulation of neutrophils. The function of NB1 gp and the role of CD177 in the pathogenesis and clinical course of polycythemia vera and essential thrombocythemia are not yet known. However, measuring neutrophil CD177 mRNA levels has become an important marker for diagnosing the myeloproliferative disorders polycythemia vera and essential thrombocythemia.

  4. Genomic organization of the mouse peroxisome proliferator-activated receptor beta/delta gene

    Larsen, Leif K; Amri, Ez-Zoubir; Mandrup, Susanne


    containing eight upstream AUG codons. We show that the presence of the 548 nt leader resulted in a low translational efficiency of the corresponding PPARbeta/delta mRNA and propose, based on structural features of the 5'-untranslated region, that translational initiation may be mediated via an internal...... gene encoding PPARbeta/delta. The gene spans approx. 41 kb and comprises 11 exons of which the six exons located in the 3'-end of the gene are included in all transcripts. Primer-extension and 5'-rapid amplification of cDNA ends experiments revealed the presence of multiple transcription start points...

  5. Downregulation of steroid receptor coactivator-2 modulates estrogen-responsive genes and stimulates proliferation of mcf-7 breast cancer cells.

    Fenne, Ingvild S; Helland, Thomas; Flågeng, Marianne H; Dankel, Simon N; Mellgren, Gunnar; Sagen, Jørn V


    The p160/Steroid Receptor Coactivators SRC-1, SRC-2/GRIP1, and SRC-3/AIB1 are important regulators of Estrogen Receptor alpha (ERα) activity. However, whereas the functions of SRC-1 and SRC-3 in breast tumourigenesis have been extensively studied, little is known about the role of SRC-2. Previously, we reported that activation of the cAMP-dependent protein kinase, PKA, facilitates ubiquitination and proteasomal degradation of SRC-2 which in turn leads to inhibition of SRC-2-coactivation of ERα and changed expression of the ERα target gene, pS2. Here we have characterized the global program of transcription in SRC-2-depleted MCF-7 breast cancer cells using short-hairpin RNA technology, and in MCF-7 cells exposed to PKA activating agents. In order to identify genes that may be regulated through PKA-induced downregulation of SRC-2, overlapping transcriptional targets in response to the respective treatments were characterized. Interestingly, we observed decreased expression of several breast cancer tumour suppressor genes (e.g., TAGLN, EGR1, BCL11b, CAV1) in response to both SRC-2 knockdown and PKA activation, whereas the expression of a number of other genes implicated in cancer progression (e.g., RET, BCAS1, TFF3, CXCR4, ADM) was increased. In line with this, knockdown of SRC-2 also stimulated proliferation of MCF-7 cells. Together, these results suggest that SRC-2 may have an antiproliferative function in breast cancer cells.

  6. Localization of the Laevigatum powdery mildew resistance gene to barley chromosome 2 by the use of RFLP markers

    Giese, H.; Holm-Jensen, A.G.; Jensen, H.P.;


    The powdery mildew disease resistance gene Ml(La) was found to belong to a locus on barely chromosome 2. We suggest that this locus be designated MlLa. Linkage analysis was carried out on 72 chromosome-doubled, spring-type progeny lines from a cross between the winter var 'Vogelsanger Gold' and t......' and the spring var 'Alf'. A map of chromosome 2 spanning 119 cM and flanked by two peroxidase gene loci was constructed. In addition to the Laevigatum resistance locus the map includes nine RFLP markers, the two peroxidase gene loci and the six-row locus in barley....

  7. Mapping of the nuclear fertility restorer gene for HL cytoplasmic male sterility in rice using microsatellite markers


    Bulked segregant analysis (BSA) of a BC1 population derived from Congguang 41A//Miyang 23/Congguang 41B was used to map the nuclear fertility restorer gene for Honglian (HL) cytoplasmic male sterility.One hundred and fifty-nine microsatellite primer pairs were screened for polymorphisms between the parents and between two bulks representing fertile and sterile plants.One microsatellite marker RM258 produced polymorphic products.The nuclear fertility restorer gene for HL cytoplasmic male sterility was mapped on chromosome 10,7.8cM from RM258.The restorer gene may be clustered on chromosome.

  8. Construction of interference vector targeting Ep-CAM gene and its effects on colorectal cancer cell proliferation

    Qi Y


    Full Text Available Yanmei Qi,1 Fengqiang Zhou,2 Lu Zhang,2 Lei Liu,2 Hong Xu,2 Huiguang Guo2 1Department of Gastroenterology, 2Department of General Surgery, Binzhou People’s Hospital, Binzhou, Shandong, People’s Republic of China Background: Prior study indicates that abnormal protein expression and functional changes in the development and progression of colorectal cancer is related to gene expression. The aim of this study was to construct an interference plasmid targeting the Ep-CAM gene and to investigate its effects on the proliferation of colorectal cancer cells.  Methods: In this study, HT-29 and HCT-116 colorectal cancer cell lines were selected as cell models. The double-stranded micro (miRNA oligo was inserted into the pcDNATM6.2-GW/EmGFPmiR vector, which is an expression of miRNA. Lipofectamine™ 2000 was used to transfer plasmid into the empty plasmid group (transfected pcDNATM6.2-GW/EmGFPmiR-neg and the interference group (transfected pcDNATM6.2-GW/EmGFPmiR-Ep-CAM-1, respectively. Meanwhile, the nontransferred HT-29 and HCT-116 acts as the blank control group. Reverse transcription polymerase chain reaction (RT-PCR was used to detect the transfection efficiency. Western blot was used to detect Ep-CAM protein expression. The cell proliferation in each group was detected by using 3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay. Results: The results indicated that the Ep-CAM messenger (mRNA expression in the interference group was lower significantly compared with that of the empty plasmid group and control group (P<0.01. Western blot analysis results showed that Ep-CAM protein expression was significantly lower in interference group compared with that of the empty plasmid group and the control group (P<0.01. MTT assay results demonstrated that the proliferation ability of cells in the interference group was significantly inhibited compared with the two other groups (P<0.05.  Conclusion: Silencing of Ep-CAM can

  9. Methylation of serum SST gene is an independent prognostic marker in colorectal cancer

    Liu, Yanqun; Chew, Min Hoe; Tham, Chee Kian; Tang, Choong Leong; Ong, Simon YK; Zhao, Yi


    There is an increasing demand for accurate prognostication for colorectal cancer (CRC). This study sought to assess prognostic potentials of methylation targets in the serum of CRC patients. A total of 165 CRC patients were enrolled in this prospective study. Promoter methylation levels of seven genes in pre-operative sera and matched tumor tissues were evaluated by quantitative methylation-specific PCR. Kaplan-Meier test, and univariate and multivariate Cox proportional hazards regression models were used for survival analyses. After a median follow-up of 56 months, 43 patients (28.7%) experienced tumor recurrence. In univariate survival analyses, serum methylation levels of SST and MAL were significantly predictive of cancer-specific death (Pcancer death and recurrence, respectively). When focusing on stage II and III patients, prognostication with serum methylated SST remained significant. Methylated SST detected in all serum samples can be traced back to the matched primary tumor tissues. We believe that methylated SST detected in the pre-operative sera of CRC patients appear to be a novel promising prognostic marker and probably can be auxiliary to tumor staging system and serum carcinoembryonic antigen towards better risk stratification.

  10. Adiposity dependent apelin gene expression: relationships with oxidative and inflammation markers.

    García-Díaz, Diego; Campión, Javier; Milagro, Fermín I; Martínez, Jose A


    It has been reported that apelin functions as an adipokine, which has been associated to obesity and insulin resistance. The objective of this study was to analyze the apelin mRNA expression in white adipose tissue (WAT) from high-fat (Cafeteria) fed rats, in order to examine potential relationships with obesity markers and other related risk factors. Animals fed on the high-fat diet during 56 days increased their body weight, total body fat and WAT depots weights when compared to controls. Apelin subcutaneous mRNA expression was higher in the Cafeteria than in the Control fed group and this increase was partially reversed by dietary vitamin C supplementation. Statistically significant associations between subcutaneous apelin gene expression and almost all the studied variables were identified, being of special interest the correlations found with serum leptin (r=0.517), liver malondialdehyde (MDA) levels (r=0.477), and leptin, IRS-3 and IL-1ra retroperitoneal mRNA expression (r=0.701; r=0.692 and r=0.561, respectively). These associations evidence a possible role for apelin in the excessive weight gain induced by high-fat feeding and increased adiposity, insulin-resistance, liver oxidative stress and inflammation.

  11. Spectinomycin resistance mutations in the rrn16 gene are new plastid markers in Medicago sativa.

    Dudas, Brigitta; Jenes, Barnabas; Kiss, Gyorgy Botond; Maliga, Pal


    We report here the isolation of spectinomycin-resistant mutants in cultured cells of Medicago sativa line RegenSY-T2. Spectinomycin induces bleaching of cultured alfalfa cells due to inhibition of protein synthesis on the prokaryotic type 70S plastid ribosomes. Spontaneous mutants resistant to spectinomycin bleaching were identified by their ability to form green shoots on plant regeneration medium containing selective spectinomycin concentrations in the range of 25-50 mg/l. Sequencing of the plastid rrn16 gene revealed that spectinomycin resistance is due to mutations in a conserved stem structure of the 16S rRNA. Resistant plants transferred to the greenhouse developed normally and produced spectinomycin-resistant seed progeny. In light of their absence in soybean, a related leguminous plant, the isolation of spectinomycin-resistant mutants in M. sativa was unexpected. The new mutations are useful for the study of plastid inheritance, as demonstrated by detection of predominantly paternal plastid inheritance in the RegenSY-T2 × Szapko57 cross, and can be used as selective markers in plastid transformation vectors to obtain cisgenic plants.

  12. 'A variant of uncertain significance' and the proliferation of human disease gene databases

    Nelson David R


    Full Text Available Abstract The rapid accumulation of mutation data has led to the creation of nearly 300 locus-specific mutation databases. These sites may contain a few dozen to almost 20,000 mutations for a given gene. Many of the mutations are uncharacterised and have no known effects on the gene product, the 'variant of uncertain significance'. Here, the statistics of mutation distribution are examined for six different gene databases: BRCA1 and BRCA2, haemoglobin-beta (HBB, HPRT1, CFTR and TP53. The percentage of all possible point mutations for a protein (the mutation space is calculated for each gene and the question 'How much mutation data is enough?' is raised.

  13. Dithizone staining of intracellular zinc: an unexpected and versatile counterscreen for auxotrophic marker genes in Saccharomyces cerevisiae.

    Daniel S Yuan

    Full Text Available Auxotrophic marker genes such as URA3, LEU2, and HIS3 in Saccharomyces cerevisiae have long been used to select cells that have been successfully transformed with recombinant DNA. A longstanding challenge in working with these genes is that counterselection procedures are often lacking. This paper describes the unexpected discovery of a simple plate assay that imparts a bright red stain to cells experiencing nutritional stress from the lack of a marker gene. The procedure specifically stains a zinc-rich vesicular compartment analogous to the zinc-rich secretory vesicles found in insulin-secreting pancreatic islet cells and glutamate-secreting neurons. Staining was greatly diminished in zap1 mutants, which lack a homeostatic activator of zinc uptake, and in cot1 zrc1 double mutants, which lack the two yeast homologs of mammalian vesicle-specific zinc export proteins. Only one of 93 strains with temperature-sensitive alleles of essential genes exhibited an increase in dithizone staining at its non-permissive temperature, indicating that staining is not simply a sign of growth-arrested or dying cells. Remarkably, the procedure works with most commonly used marker genes, highlights subtle defects, uses no reporter constructs or expensive reagents, requires only a few hours of incubation, yields visually striking results without any instrumentation, and is not toxic to the cells. Many potential applications exist for dithizone staining, both as a versatile counterscreen for auxotrophic marker genes and as a powerful new tool for the genetic analysis of a biomedically important vesicular organelle.

  14. Differential Regulation of Gene Expression of Alveolar Epithelial Cell Markers in Human Lung Adenocarcinoma-Derived A549 Clones

    Hiroshi Kondo


    Full Text Available Stem cell therapy appears to be promising for restoring damaged or irreparable lung tissue. However, establishing a simple and reproducible protocol for preparing lung progenitor populations is difficult because the molecular basis for alveolar epithelial cell differentiation is not fully understood. We investigated an in vitro system to analyze the regulatory mechanisms of alveolus-specific gene expression using a human alveolar epithelial type II (ATII cell line, A549. After cloning A549 subpopulations, each clone was classified into five groups according to cell morphology and marker gene expression. Two clones (B7 and H12 were further analyzed. Under serum-free culture conditions, surfactant protein C (SPC, an ATII marker, was upregulated in both H12 and B7. Aquaporin 5 (AQP5, an ATI marker, was upregulated in H12 and significantly induced in B7. When the RAS/MAPK pathway was inhibited, SPC and thyroid transcription factor-1 (TTF-1 expression levels were enhanced. After treatment with dexamethasone (DEX, 8-bromoadenosine 3′5′-cyclic monophosphate (8-Br-cAMP, 3-isobutyl-1-methylxanthine (IBMX, and keratinocyte growth factor (KGF, surfactant protein B and TTF-1 expression levels were enhanced. We found that A549-derived clones have plasticity in gene expression of alveolar epithelial differentiation markers and could be useful in studying ATII maintenance and differentiation.

  15. Cancer specificity of promoters of the genes involved in cell proliferation control.

    Kashkin, K N; Chernov, I P; Stukacheva, E A; Kopantzev, E P; Monastyrskaya, G S; Uspenskaya, N Ya; Sverdlov, E D


    Core promoters with adjacent regions of the human genes CDC6, POLD1, CKS1B, MCM2, and PLK1 were cloned into a pGL3 vector in front of the Photinus pyrails gene Luc in order to study the tumor specificity of the promoters. The cloned promoters were compared in their ability to direct luciferase expression in different human cancer cells and in normal fibroblasts. The cancer-specific promoter BIRC5 and non-specific CMV immediately early gene promoter were used for comparison. All cloned promoters were shown to be substantially more active in cancer cells than in fibroblasts, while the PLK1 promoter was the most cancer-specific and promising one. The specificity of the promoters to cancer cells descended in the series PLK1, CKS1B, POLD1, MCM2, and CDC6. The bidirectional activity of the cloned CKS1B promoter was demonstrated. It apparently directs the expression of the SHC1 gene, which is located in a "head-to-head" position to the CKS1B gene in the human genome. This feature should be taken into account in future use of the CKS1B promoter. The cloned promoters may be used in artificial genetic constructions for cancer gene therapy.

  16. Retransformation of marker-free potato for enhanced resistance against fungal pathogens by pyramiding chitinase and wasabi defensin genes.

    Khan, Raham Sher; Darwish, Nader Ahmed; Khattak, Bushra; Ntui, Valentine Otang; Kong, Kynet; Shimomae, Kazuki; Nakamura, Ikuo; Mii, Masahiro


    Multi-auto-transformation vector system has been one of the strategies to produce marker-free transgenic plants without using selective chemicals and plant growth regulators and thus facilitating transgene stacking. In the study reported here, retransformation was carried out in marker-free transgenic potato CV. May Queen containing ChiC gene (isolated from Streptomyces griseus strain HUT 6037) with wasabi defensin (WD) gene (isolated from Wasabia japonica) to pyramid the two disease resistant genes. Molecular analyses of the developed shoots confirmed the existence of both the genes of interest (ChiC and WD) in transgenic plants. Co-expression of the genes was confirmed by RT-PCR, northern blot, and western blot analyses. Disease resistance assay of in vitro plants showed that the transgenic lines co-expressing both the ChiC and WD genes had higher resistance against the fungal pathogens, Fusarium oxysporum (Fusarium wilt) and Alternaria solani (early blight) compared to the non-transformed control and the transgenic lines expressing either of the ChiC or WD genes. The disease resistance potential of the transgenic plants could be increased by transgene stacking or multiple transformations.

  17. A computational procedure for functional characterization of potential marker genes from molecular data: Alzheimer's as a case study

    Barla Annalisa


    Full Text Available Abstract Background A molecular characterization of Alzheimer's Disease (AD is the key to the identification of altered gene sets that lead to AD progression. We rely on the assumption that candidate marker genes for a given disease belong to specific pathogenic pathways, and we aim at unveiling those pathways stable across tissues, treatments and measurement systems. In this context, we analyzed three heterogeneous datasets, two microarray gene expression sets and one protein abundance set, applying a recently proposed feature selection method based on regularization. Results For each dataset we identified a signature that was successively evaluated both from the computational and functional characterization viewpoints, estimating the classification error and retrieving the most relevant biological knowledge from different repositories. Each signature includes genes already known to be related to AD and genes that are likely to be involved in the pathogenesis or in the disease progression. The integrated analysis revealed a meaningful overlap at the functional level. Conclusions The identification of three gene signatures showing a relevant overlap of pathways and ontologies, increases the likelihood of finding potential marker genes for AD.

  18. Gene Expression Profiling Soybean Stem Tissue Early Response to Sclerotinia sclerotiorum and In Silico Mapping in Relation to Resistance Markers

    Bernarda Calla


    Full Text Available White mold, caused by (Lib. de Bary, can be a serious disease of crops grown under cool, moist environments. In many plants, such as soybean [ (L. Merr.], complete genetic resistance does not exist. To identify possible genes involved in defense against this pathogen, and to determine possible physiological changes that occur during infection, a microarray screen was conducted using stem tissue to evaluate changes in gene expression between partially resistant and susceptible soybean genotypes at 8 and 14 hours post inoculation. RNA from 15 day-old inoculated plants was labeled and hybridized to soybean cDNA microarrays. ANOVA identified 1270 significant genes from the comparison between time points and 105 genes from the comparison between genotypes. Selected genes were classified into functional categories. The analyses identified changes in cell-wall composition and signaling pathways, as well as suggesting a role for anthocyanin and anthocyanidin synthesis in the defense against . In-silico mapping of both the differentially expressed transcripts and of public markers associated with partial resistance to white mold, provided evidence of several differentially expressed genes being closely positioned to white mold resistance markers, with the two most promising genes encoding a PR-5 and anthocyanidin synthase.

  19. Identification of potential transcriptomic markers in developing ankylosing spondylitis: a meta-analysis of gene expression profiles.

    Fang, Fang; Pan, Jian; Xu, Lixiao; Li, Gang; Wang, Jian


    The goal of this study was to identify potential transcriptomic markers in developing ankylosing spondylitis by a meta-analysis of multiple public microarray datasets. Using the INMEX (integrative meta-analysis of expression data) program, we performed the meta-analysis to identify consistently differentially expressed (DE) genes in ankylosing spondylitis and further performed functional interpretation (gene ontology analysis and pathway analysis) of the DE genes identified in the meta-analysis. Three microarray datasets (26 cases and 29 controls in total) were collected for meta-analysis. 905 consistently DE genes were identified in ankylosing spondylitis, among which 482 genes were upregulated and 423 genes were downregulated. The upregulated gene with the smallest combined rank product (RP) was GNG11 (combined RP=299.64). The downregulated gene with the smallest combined RP was S100P (combined RP=335.94). In the gene ontology (GO) analysis, the most significantly enriched GO term was "immune system process" (P=3.46×10(-26)). The most significant pathway identified in the pathway analysis was antigen processing and presentation (P=8.40×10(-5)). The consistently DE genes in ankylosing spondylitis and biological pathways associated with those DE genes identified provide valuable information for studying the pathophysiology of ankylosing spondylitis.

  20. Identification of Potential Transcriptomic Markers in Developing Ankylosing Spondylitis: A Meta-Analysis of Gene Expression Profiles

    Fang, Fang; Pan, Jian; Xu, Lixiao; Li, Gang; Wang, Jian


    The goal of this study was to identify potential transcriptomic markers in developing ankylosing spondylitis by a meta-analysis of multiple public microarray datasets. Using the INMEX (integrative meta-analysis of expression data) program, we performed the meta-analysis to identify consistently differentially expressed (DE) genes in ankylosing spondylitis and further performed functional interpretation (gene ontology analysis and pathway analysis) of the DE genes identified in the meta-analysis. Three microarray datasets (26 cases and 29 controls in total) were collected for meta-analysis. 905 consistently DE genes were identified in ankylosing spondylitis, among which 482 genes were upregulated and 423 genes were downregulated. The upregulated gene with the smallest combined rank product (RP) was GNG11 (combined RP = 299.64). The downregulated gene with the smallest combined RP was S100P (combined RP = 335.94). In the gene ontology (GO) analysis, the most significantly enriched GO term was “immune system process” (P = 3.46 × 10−26). The most significant pathway identified in the pathway analysis was antigen processing and presentation (P = 8.40 × 10−5). The consistently DE genes in ankylosing spondylitis and biological pathways associated with those DE genes identified provide valuable information for studying the pathophysiology of ankylosing spondylitis. PMID:25688367

  1. Fate and proliferation of typical antibiotic resistance genes in five full-scale pharmaceutical wastewater treatment plants

    Wang, Jilu [College of Environmental Science and Engineering, Ministry of Education Key Laboratory of Pollution Processes and Environmental Criteria, Nankai University, Tianjin 300071 (China); Mao, Daqing, E-mail: [School of Environmental Science and Engineering, Tianjin University, Tianjin 300072 (China); Mu, Quanhua [College of Environmental Science and Engineering, Ministry of Education Key Laboratory of Pollution Processes and Environmental Criteria, Nankai University, Tianjin 300071 (China); Luo, Yi, E-mail: [College of Environmental Science and Engineering, Ministry of Education Key Laboratory of Pollution Processes and Environmental Criteria, Nankai University, Tianjin 300071 (China)


    This study investigated the characteristics of 10 subtypes of antibiotic resistance genes (ARGs) for sulfonamide, tetracycline, β-lactam and macrolide resistance and the class 1 integrase gene (intI1). In total, these genes were monitored in 24 samples across each stage of five full-scale pharmaceutical wastewater treatment plants (PWWTPs) using qualitative and real-time quantitative polymerase chain reactions (PCRs). The levels of typical ARG subtypes in the final effluents ranged from (2.08 ± 0.16) × 10{sup 3} to (3.68 ± 0.27) × 10{sup 6} copies/mL. The absolute abundance of ARGs in effluents accounted for only 0.6%–59.8% of influents of the five PWWTPs, while the majority of the ARGs were transported to the dewatered sludge with concentrations from (9.38 ± 0.73) × 10{sup 7} to (4.30 ± 0.81) × 10{sup 10} copies/g dry weight (dw). The total loads of ARGs discharged through dewatered sludge was 7–308 folds higher than that in the raw influents and 16–638 folds higher than that in the final effluents. The proliferation of ARGs mainly occurs in the biological treatment processes, such as conventional activated sludge, cyclic activated sludge system (CASS) and membrane bio-reactor (MBR), implying that significant replication of certain subtypes of ARGs may be attributable to microbial growth. High concentrations of antibiotic residues (ranging from 0.14 to 92.2 mg/L) were detected in the influents of selected wastewater treatment systems and they still remain high residues in the effluents. Partial correlation analysis showed significant correlations between the antibiotic concentrations and the associated relative abundance of ARG subtypes in the effluent. Although correlation does not prove causation, this study demonstrates that in addition to bacterial growth, the high antibiotic residues within the pharmaceutical WWTPs may influence the proliferation and fate of the associated ARG subtypes. - Highlights: • The ARGs in final discharges were 7

  2. Epigenetic transcriptional regulation of the growth arrest-specific gene 1 (Gas1 in hepatic cell proliferation at mononucleosomal resolution.

    Natalia Sacilotto

    Full Text Available BACKGROUND: Gas1 (growth arrest-specific 1 gene is known to inhibit cell proliferation in a variety of models, but its possible implication in regulating quiescence in adult tissues has not been examined to date. The knowledge of how Gas1 is regulated in quiescence may contribute to understand the deregulation occurring in neoplastic diseases. METHODOLOGY/PRINCIPAL FINDINGS: Gas1 expression has been studied in quiescent murine liver and during the naturally synchronized cell proliferation after partial hepatectomy. Chromatin immunoprecipitation at nucleosomal resolution (Nuc-ChIP has been used to carry out the study preserving the in vivo conditions. Transcription has been assessed at real time by quantifying the presence of RNA polymerase II in coding regions (RNApol-ChIP. It has been found that Gas1 is expressed not only in quiescent liver but also at the cell cycle G(1/S transition. The latter expression peak had not been previously reported. Two nucleosomes, flanking a nucleosome-free region, are positioned close to the transcription start site. Both nucleosomes slide in going from the active to the inactive state and vice versa. Nuc-ChIP analysis of the acquisition of histone epigenetic marks show distinctive features in both active states: H3K9ac and H3K4me2 are characteristic of transcription in G(0 and H4R3me2 in G(1/S transition. Sequential-ChIP analysis revealed that the "repressing" mark H3K9me2 colocalize with several "activating" marks at nucleosome N-1 when Gas1 is actively transcribed suggesting a greater plasticity of epigenetic marks than proposed until now. The recruitment of chromatin-remodeling or modifying complexes also displayed distinct characteristics in quiescence and the G(1/S transition. CONCLUSIONS/SIGNIFICANCE: The finding that Gas1 is transcribed at the G(1/S transition suggests that the gene may exert a novel function during cell proliferation. Transcription of this gene is modulated by specific "activating" and

  3. Effects of salvianolic acid-A on NIH/3T3 fibroblast proliferation, collagen synthesis and gene expression

    Liu, Cheng-Hai; Hu, Yi-Yang; Wang, Xiao-Ling; Xu, Lie-Ming; Liu, Ping


    AIM: To investigate the mechanisms of salvianolic acid A (SA-A) against liver fibrosis in vitro. METHODS: NIH/3T3 fibroblasts were cultured routinely, and incubated with 10-4 mol/L-10-7 mol/L SA-A for 22 h. The cell viability was assayed by [3H]proline incorporation, cell proliferation by [3H]TdR incorporation, cell collagen synthetic rate was measured with [3H]proline impulse and collagenase digestion method. The total RNA was prepared from the control cells and the drug treated cells respectively, and α (1) I pro-collagen mRNA expression was semi-quantitatively analyzed with RT-PCR. RESULTS: 10-4 mol/L SA-A decreased cell viability and exerted some cytotoxiciy, while 10-5 mol/L-10-7 mol/L SA-A did not affect cell viability, but inhibited cell proliferation significantly, and 10-6 mol/L SA-A had the best effect on cell viability among these concentrations of drugs. 10-5 mol/L-10-6 mol/L SA-A inhibited intracellular collagen synthetic rate, but no significant influence on extracellular collagen secretion. Both 10-5 mol/L and 10-6 mol/L SA-A could decrease α (1) I pro-collagen mRNA expression remarkably. CONCLUSION: SA-A had potent action against liver fibrosis. It inhibited NIH/3T3 fibroblast proliferation, intracellular collagen synthetic rate and type I pro-collagen gene expression, which may be one of the main mechanisms of the drug. PMID:11819598

  4. Autism and genetics: Clinical approach and association study with two markers of HRAS gene

    Herault, J.; Petit, E.; Cherpi, C. [Laboratoire de Biochimie Medicale, Tours (France)] [and others


    Twin studies and familial aggregation studies indicate that genetic factors could play a role in infantile autism. In an earlier study, we identified a possible positive association between autism and a c-Harvey-ras (HRAS) oncogene marker at the 3{prime} end of the coding region. In an attempt to confirm this finding, we studied a larger population, well-characterized clinically and genetically. We report a positive association between autism and two HRAS markers, the 3{prime} marker used in the initial study and an additional marker in exon 1. 46 refs., 1 fig., 2 tabs.

  5. Inhibition of Proliferation of Human Hela Cells by Small Interference RNA against Pokemon Gene

    DENG Yi-jing; NI Bing; JIANG Man; YANG Di; LI Fan; WU Yu-zhang


    Objective:The transcriptional repressor Pokemon(encoded by the Zbtb7 gene)is a critical factor in oncogenesis.Pokemon overexpression leads to overt oncogenic transformation both in vitro and in vivo in transgenic mice. The objective of this study was to investigate the effect of retrovirus expressing the siRNA targeting Pokemon in human cervical cancer cells. Methods:We constructed and identified the recombinant retrovirus particle expressing siRNA of Pokemon gene,and then testified the suppression of recombinant plasmid and evaluated the gene-silencing effect. Results:We got the positive evaluation from colony forming experiment we found that the retrovirus expressing siRNA targeting Pokemon had repressing effect. Conclusion:Our work provides basis for the study of suppression effect of retrovirus in vivo and the design of the target-complex.

  6. Establishing quiescence in human bone marrow stem cells leads to enhanced osteoblast marker expression

    Harkness, Linda; Rumman, Mohammad; Kassem, Moustapha;

    expression profiling of the cells demonstrated down-regulation of cyclin (CCNA2, CCND1, CCNE1, CCNB1) and proliferation markers (Ki67) markers during G0 and up-regulation of the osteogenic genes RUNX2 and OPN. RT-PCR analysis of osteogenic differentiation of cells post G0 demonstrated an increase...

  7. Standardization of Gene Expression Quantification by Absolute Real-Time qRT-PCR System Using a Single Standard for Marker and Reference Genes

    Yi-Hong Zhou


    Full Text Available In the last decade, genome-wide gene expression data has been collected from a large number of cancer specimens. In many studies utilizing either microarray-based or knowledge-based gene expression profiling, both the validation of candidate genes and the identification and inclusion of biomarkers in prognosis-modeling has employed real-time quantitative PCR on reverse transcribed mRNA (qRT-PCR because of its inherent sensitivity and quantitative nature. In qRT-PCR data analysis, an internal reference gene is used to normalize the variation in input sample quantity. The relative quantification method used in current real-time qRT-PCR analysis fails to ensure data comparability pivotal in identification of prognostic biomarkers. By employing an absolute qRT-PCR system that uses a single standard for marker and reference genes (SSMR to achieve absolute quantification, we showed that the normalized gene expression data is comparable and independent of variations in the quantities of sample as well as the standard used for generating standard curves. We compared two sets of normalized gene expression data with same histological diagnosis of brain tumor from two labs using relative and absolute real-time qRT-PCR. Base-10 logarithms of the gene expression ratio relative to ACTB were evaluated for statistical equivalence between tumors processed by two different labs. The results showed an approximate comparability for normalized gene expression quantified using a SSMR-based qRT-PCR. Incomparable results were seen for the gene expression data using relative real-time qRT-PCR, due to inequality in molar concentration of two standards for marker and reference genes. Overall results show that SSMR-based real-time qRT-PCR ensures comparability of gene expression data much needed in establishment of prognostic/predictive models for cancer patients—a process that requires large sample sizes by combining independent sets of data.

  8. Genes conserved in bilaterians but jointly lost with Myc during nematode evolution are enriched in cell proliferation and cell migration functions.

    Erives, Albert J


    Animals use a stereotypical set of developmental genes to build body architectures of varying sizes and organizational complexity. Some genes are critical to developmental patterning, while other genes are important to physiological control of growth. However, growth regulator genes may not be as important in small-bodied "micro-metazoans" such as nematodes. Nematodes use a simplified developmental strategy of lineage-based cell fate specifications to produce an adult bilaterian body composed of a few hundreds of cells. Nematodes also lost the MYC proto-oncogenic regulator of cell proliferation. To identify additional regulators of cell proliferation that were lost with MYC, we computationally screened and determined 839 high-confidence genes that are conserved in bilaterians/lost in nematodes (CIBLIN genes). We find that 30 % of all CIBLIN genes encode transcriptional regulators of cell proliferation, epithelial-to-mesenchyme transitions, and other processes. Over 50 % of CIBLIN genes are unnamed genes in Drosophila, suggesting that there are many understudied genes. Interestingly, CIBLIN genes include many Myc synthetic lethal (MycSL) hits from recent screens. CIBLIN genes include key regulators of heparan sulfate proteoglycan (HSPG) sulfation patterns, and lysyl oxidases involved in cross-linking and modification of the extracellular matrix (ECM). These genes and others suggest the CIBLIN repertoire services critical functions in ECM remodeling and cell migration in large-bodied bilaterians. Correspondingly, CIBLIN genes are co-expressed with Myc in cancer transcriptomes, and include a preponderance of known determinants of cancer progression and tumor aggression. We propose that CIBLIN gene research can improve our understanding of regulatory control of cellular growth in metazoans.

  9. De novo characterization of Larimichthys crocea transcriptome for growth-/immune-related gene identification and massive microsatellite (SSR) marker development

    Han, Zhaofang; Xiao, Shijun; Liu, Xiande; Liu, Yang; Li, Jiakai; Xie, Yangjie; Wang, Zhi Yong


    The large yellow croaker, Larimichthys crocea is an important marine fish in China with a high economic value. In the last decade, the stock conservation and aquaculture industry of this species have been facing severe challenges because of wild population collapse and degeneration of important economic traits. However, genes contributing to growth and immunity in L. crocea have not been thoroughly analyzed, and available molecular markers are still not sufficient for genetic resource management and molecular selection. In this work, we sequenced the transcriptome in L. crocea liver tissue with a Roche 454 sequencing platform and assembled the transcriptome into 93 801 transcripts. Of them, 38 856 transcripts were successfully annotated in nt, nr, Swiss-Prot, InterPro, COG, GO and KEGG databases. Based on the annotation information, 3 165 unigenes related to growth and immunity were identified. Additionally, a total of 6 391 simple sequence repeats (SSRs) were identified from the transcriptome, among which 4 498 SSRs had enough flanking regions to design primers for polymerase chain reactions (PCR). To access the polymorphism of these markers, 30 primer pairs were randomly selected for PCR amplification and validation in 30 individuals, and 12 primer pairs (40.0%) exhibited obvious length polymorphisms. This work applied RNA-Seq to assemble and analyze a live transcriptome in L. crocea. With gene annotation and sequence information, genes related to growth and immunity were identified and massive SSR markers were developed, providing valuable genetic resources for future gene functional analysis and selective breeding of L. crocea.

  10. De novo characterization of Larimichthys crocea transcriptome for growth-/immune-related gene identification and massive microsatellite (SSR) marker development

    Han, Zhaofang; Xiao, Shijun; Liu, Xiande; Liu, Yang; Li, Jiakai; Xie, Yangjie; Wang, Zhiyong


    The large yellow croaker, Larimichthys crocea is an important marine fish in China with a high economic value. In the last decade, the stock conservation and aquaculture industry of this species have been facing severe challenges because of wild population collapse and degeneration of important economic traits. However, genes contributing to growth and immunity in L. crocea have not been thoroughly analyzed, and available molecular markers are still not sufficient for genetic resource management and molecular selection. In this work, we sequenced the transcriptome in L. crocea liver tissue with a Roche 454 sequencing platform and assembled the transcriptome into 93 801 transcripts. Of them, 38 856 transcripts were successfully annotated in nt, nr, Swiss-Prot, InterPro, COG, GO and KEGG databases. Based on the annotation information, 3 165 unigenes related to growth and immunity were identified. Additionally, a total of 6 391 simple sequence repeats (SSRs) were identified from the transcriptome, among which 4 498 SSRs had enough flanking regions to design primers for polymerase chain reactions (PCR). To access the polymorphism of these markers, 30 primer pairs were randomly selected for PCR amplification and validation in 30 individuals, and 12 primer pairs (40.0%) exhibited obvious length polymorphisms. This work applied RNA-Seq to assemble and analyze a live transcriptome in L. crocea. With gene annotation and sequence information, genes related to growth and immunity were identified and massive SSR markers were developed, providing valuable genetic resources for future gene functional analysis and selective breeding of L. crocea.

  11. Breeding of Selectable Marker-Free Transgenic Rice Lines Containing AP1 Gene with Enhanced Disease Resistance

    YU Heng-xiu; LIU Qiao-quan; WANG Ling; ZHAO Zhi-peng; XU Li; HUANG Ben-li; GONG Zhi-yun; TANG Shu-zhu; GU Ming-hong


    In order to obtain marker-free transgenic rice with improved disease resistance, the AP1 gene of Capsicum annuum and hygromycin-resistance gene (HPT) were cloned into the two separate T-DNA regions of the binary vector pSB 130,respectively, and introduced into the calli derived from the immature seeds of two elite japonica rice varieties, Guangling Xiangjing and Wuxiangjing 9, mediated by Agrobacterium-mediated transformation. Many cotransgenic rice lines containing both the AP1 gene and the marker gene were regenerated and the integration of both transgenes in the transgenic rice plants was confirmed by either PCR or Southern blotting technique. Several selectable marker-free transgenic rice plants were subsequently obtained from the progeny of the cotransformants, and confirmed by both PCR and Southern blotting analysis. These transgenic rice lines were tested in the field and their resistance to disease was c arefully investigated, the results showed that after inoculation the resistance to either bacterial blight or sheath blight of the selected transgenic lines was improved when compared with those of wild type.

  12. Characterization of the miiuy croaker (Miichthys miiuy transcriptome and development of immune-relevant genes and molecular markers.

    Rongbo Che

    Full Text Available BACKGROUND: The miiuy croaker (Miichthys miiuy is an important species of marine fish that supports capture fisheries and aquaculture. At present commercial scale aquaculture of this species is limited due to diseases caused by pathogens and parasites which restrict production and limit commercial value. The lack of transcriptomic and genomic information for the miiuy croaker limits the ability of researchers to study the pathogenesis and immune system of this species. In this study we constructed a cDNA library from liver, spleen and kidney which was sequenced using Illumina paired-end sequencing to enable gene discovery and molecular marker development. PRINCIPAL FINDINGS: In our study, a total of 69,071 unigenes with an average length of 572 bp were obtained. Of these, 45,676 (66.13% were successfully annotated in public databases. The unigenes were also annotated with Gene Ontology, Clusters of Orthologous Groups and KEGG pathways. Additionally, 498 immune-relevant genes were identified and classified. Furthermore, 14,885 putative simple sequence repeats (cSSRs and 8,510 putative single nucleotide polymorphisms (SNPs were identified from the 69,071 unigenes. CONCLUSION: The miiuy croaker (Miichthys miiuy transcriptome data provides a large resource to identify new genes involved in many processes including those involved in the response to pathogens and diseases. Furthermore, the thousands of potential cSSR and SNP markers found in this study are important resources with respect to future development of molecular marker assisted breeding programs for the miiuy croaker.

  13. Novel recombinant binary vectors harbouring Basta (bar) gene as a plant selectable marker for genetic transformation of plants.

    Nada, Reham M


    Genetic transformation is one of the most widely used technique in crop improvement. However, most of the binary vectors used in this technique, especially cloning based, contain antibiotic genes as selection marker that raise serious consumer and environmental concerns; moreover, they could be transferred to non-target hosts with deleterious effects. Therefore, the goal of this study was reconstruction of the widely used pBI121 binary vector by substituting the harmful antibiotic selection marker gene with a less-harmful selection marker, Basta (herbicide resistance gene). The generated vectors were designated as pBI121NB and pBI121CB, in which Basta gene was expressed under the control of Nos or CaMV 35S promoter, respectively. The successful integration of the new inserts into both the vectors was confirmed by PCR, restriction digestion and sequencing. Both these vectors were used in transforming Arabidopsis, Egyptian wheat and barley varieties using LBA4404 and GV3101 Agrobacterium strains. The surfactant Tween-20 resulted in an efficient transformation and the number of Arabidopsis transformants was about 6-9 %. Soaked seeds of wheat and barley were transformed with Agrobacterium to introduce the bacteria to the growing shoot apices. The percentage of transgenic lines was around 16-17 and 14-15 % for wheat and barley, respectively. The quantitative studies presented in this work showed that both LBA4404 and GV3101 strains were suitable for transforming Egyptian wheat and barley.

  14. Constitutive expression of Wnt/β-catenin target genes promotes proliferation and invasion of liver cancer stem cells



    Wnt/β-catenin is an important signaling pathways involved in the tumorgenesis, progression and maintenance of cancer stem cells (CSCs). In the present study, the role of Wnt/β-catenin signaling in CSC-mediated tumorigenesis and invasion in liver CSCs was investigated. A small population of cancer stem-like side population (SP) cells (3.6%) from liver cancer samples were identified. The cells were highly resistant to drug treatment due to the enhanced expression of drug efflux pumps, such as ABC subfamily G member 2, multidrug resistance protein 1 and ATP-binding cassette subfamily B member 5. Furthermore, using TOPflash and reverse transcription-quantitative polymerase chain reaction analysis, Wnt/β-catenin signaling and the transcriptional regulation of Wnt/β-catenin target genes including dickkopf Wnt signaling pathway inhibitor 1, axis inhibition protein 2 and cyclin D1 were observed to be markedly upregulated in liver cancer SP cells. As a consequence, SP cells possessed infinite cell proliferation potential and the ability to generating tumor spheres. In addition, upon reducing Wnt/β-catenin signaling, the rates of proliferation, tumor sphere formation and tumor invasion of SP cells were markedly reduced. Therefore, these data suggest that Wnt/β-catenin signaling is a potential therapeutic target to reduce CSC-mediated tumorigenicity and invasion in liver cancer. PMID:26956539

  15. Use of Molecular Imaging Markers of Glycolysis, Hypoxia and Proliferation (18F-FDG, 64Cu-ATSM and 18F-FLT) in a Dog with Fibrosarcoma

    Zornhagen, Kamilla; Clausen, Malene; Hansen, Anders Elias


    Glycolysis, hypoxia, and proliferation are important factors in the tumor microenvironment contributing to treatment-resistant aggressiveness. Imaging these factors using combined functional positron emission tomography and computed tomography can potentially guide diagnosis and management...

  16. A high efficiency gene disruption strategy using a positive-negative split selection marker and electroporation for Fusarium oxysporum.

    Liang, Liqin; Li, Jianqiang; Cheng, Lin; Ling, Jian; Luo, Zhongqin; Bai, Miao; Xie, Bingyan


    The Fusarium oxysporum species complex consists of fungal pathogens that cause serial vascular wilt disease on more than 100 cultivated species throughout the world. Gene function analysis is rapidly becoming more and more important as the whole-genome sequences of various F. oxysporum strains are being completed. Gene-disruption techniques are a common molecular tool for studying gene function, yet are often a limiting step in gene function identification. In this study we have developed a F. oxysporum high-efficiency gene-disruption strategy based on split-marker homologous recombination cassettes with dual selection and electroporation transformation. The method was efficiently used to delete three RNA-dependent RNA polymerase (RdRP) genes. The gene-disruption cassettes of three genes can be constructed simultaneously within a short time using this technique. The optimal condition for electroporation is 10μF capacitance, 300Ω resistance, 4kV/cm field strength, with 1μg of DNA (gene-disruption cassettes). Under these optimal conditions, we were able to obtain 95 transformants per μg DNA. And after positive-negative selection, the transformants were efficiently screened by PCR, screening efficiency averaged 85%: 90% (RdRP1), 85% (RdRP2) and 77% (RdRP3). This gene-disruption strategy should pave the way for high throughout genetic analysis in F. oxysporum. Copyright © 2014 Elsevier GmbH. All rights reserved.

  17. Bmi1 gene silencing inhibits the proliferation and invasiveness of human hepatocellular carcinoma cells and increases their sensitivity to 5-fluorouracil.

    Zhang, Rui; Xu, Lei-Bo; Yue, Xiu-Jing; Yu, Xian-Huan; Wang, Jie; Liu, Chao


    The Bmi1 gene has been reported to play important roles in cancer initiation and progression. The aim of this study was to investigate the effects of RNA interference (RNAi)-mediated silencing of Bmi1 gene expression on the proliferation and invasiveness of hepatocellular carcinoma (HCC) cells and on the efficacy of chemotherapy in HCC patients. The Bmi1 gene was silenced by Bmi1-siRNA (small interfering RNA) in the human HCC cell lines HepG2 and Bel-7402, and the gene expression levels were assayed by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting. The proliferation and migration of Bmi1-silenced tumor cells and their sensitivity to 5-FU treatment were determined by Cell Counting Kit-8 (CCK-8), transwell assays and 4',6-diamidino-2-phenylindole (DAPI) staining and flow cytometry, respectively. Bmi1-siRNA inhibited the Bmi1 expression at both the mRNA and protein levels in HCC cells. Proliferation and migration of HCC cells treated with Bmi1-siRNA was significantly lower compared to that of the control cells. Moreover, Bmi1 gene silencing increased the percentage of apoptotic cells treated by 5-FU and decreased the IC50 values of 5-FU to a greater extent. Downregulation of the Bmi1 gene by RNAi can inhibit the proliferation and invasivesness of HCC cells and increase their sensitivity to 5-FU treatment.

  18. Heat-shock-mediated elimination of the nptII marker gene in transgenic apple (Malus×domestica Borkh.).

    Herzog, Katja; Flachowsky, Henryk; Deising, Holger B; Hanke, Magda-Viola


    Production of marker-free genetically modified (GM) plants is one of the major challenges of molecular fruit breeding. Employing clean vector technologies, allowing the removal of undesired DNA sequences from GM plants, this goal can be achieved. The present study describes the establishment of a clean vector system in apple Malus×domestica Borkh., which is based on the use of the neomycin phosphotransferase II gene (nptII) as selectable marker gene and kanamycin/paramomycin as selective agent. The nptII gene can be removed after selection of GM shoots via site-specific excision mediated by heat-shock-inducible expression of the budding yeast FLP recombinase driven by the soybean Gmhsp17.5-E promoter. We created a monitoring vector containing the nptII and the flp gene as a box flanked by two direct repeats of the flp recognition target (FRT) sites. The FRT-flanked box separates the gusA reporter gene from the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter. Consequently, GUS expression does only occur after elimination of the FRT-flanked box. Transformation experiments using the monitoring vector resulted in a total of nine transgenic lines. These lines were investigated for transgenicity by PCR, RT-PCR and Southern hybridization. Among different temperature regimes tested, exposure to 42 °C for 3.5 to 4h led to efficient induction of FLP-mediated recombination and removal of the nptII marker gene. A second round of shoot regeneration from leaf explants led to GM apple plants completely free of the nptII gene.

  19. Molecular Tagging and Mapping of Quantitative Trait Loci for Lint Percentage and Morphological Marker Genes in Upland Cotton


    Using 219 F2 individuals developed by crossing the genetic standard line TM-1 and the multiple dominant marker line T586 in Gossypium hirsutum L., a genetic linkage map with 19 linkage groups was constructed based on simple sequence repeat (SSR) markers. Compared with our tetraploid backboned molecular genetic map from a (TM-1xHai 7124)xTM-1 BC1 population, 17 of the 19 linkage groups were combined and anchored to 12 chromosomes (sub-genomes). Of these groups, four morphological marker genes in T586 had been mapped into the molecular linkage map. Meanwhile, three quantitative trait loci for lint percentage were tagged and mapped separately on the A03 linkage group and chromosome 6.

  20. A Self-deleting Cre-lox-ermAM Cassette, CHESHIRE, for marker-less gene deletion in Streptococcus pneumoniae

    Weng, Liming; Biswas, Indranil; Morrison, Donald A.


    Although targeted mutagenesis of Streptococcus pneumoniae is readily accomplished with the aid of natural genetic transformation and chimeric donor DNA constructs assembled in vitro, the drug resistance markers often employed for selection of recombinant products can themselves be undesirable by-products of the genetic manipulation. A new cassette carrying the erythromycin-resistance marker ermAM is described that can be used as a temporary marker for selection of desired recombinants. The cassette may subsequently be removed at will by virtue of an embedded fucose-regulated Cre recombinase gene and terminal lox66 and lox71 Cre recognition sites, with retention of 34 bp from the cassette as an inert residual double-mutant lox72 site. PMID:19850089

  1. Transcriptome sequencing of different narrow-leafed lupin tissue types provides a comprehensive uni-gene assembly and extensive gene-based molecular markers

    Kamphuis, Lars G; Hane, James K; Nelson, Matthew N; Gao, Lingling; Atkins, Craig A; Singh, Karam B


    Narrow-leafed lupin (NLL; Lupinus angustifolius L.) is an important grain legume crop that is valuable for sustainable farming and is becoming recognized as a human health food. NLL breeding is directed at improving grain production, disease resistance, drought tolerance and health benefits. However, genetic and genomic studies have been hindered by a lack of extensive genomic resources for the species. Here, the generation, de novo assembly and annotation of transcriptome datasets derived from five different NLL tissue types of the reference accession cv. Tanjil are described. The Tanjil transcriptome was compared to transcriptomes of an early domesticated cv. Unicrop, a wild accession P27255, as well as accession 83A:476, together being the founding parents of two recombinant inbred line (RIL) populations. In silico predictions for transcriptome-derived gene-based length and SNP polymorphic markers were conducted and corroborated using a survey assembly sequence for NLL cv. Tanjil. This yielded extensive indel and SNP polymorphic markers for the two RIL populations. A total of 335 transcriptome-derived markers and 66 BAC-end sequence-derived markers were evaluated, and 275 polymorphic markers were selected to genotype the reference NLL 83A:476 × P27255 RIL population. This significantly improved the completeness, marker density and quality of the reference NLL genetic map. PMID:25060816

  2. Transcriptome sequencing of different narrow-leafed lupin tissue types provides a comprehensive uni-gene assembly and extensive gene-based molecular markers.

    Kamphuis, Lars G; Hane, James K; Nelson, Matthew N; Gao, Lingling; Atkins, Craig A; Singh, Karam B


    Narrow-leafed lupin (NLL; Lupinus angustifolius L.) is an important grain legume crop that is valuable for sustainable farming and is becoming recognized as a human health food. NLL breeding is directed at improving grain production, disease resistance, drought tolerance and health benefits. However, genetic and genomic studies have been hindered by a lack of extensive genomic resources for the species. Here, the generation, de novo assembly and annotation of transcriptome datasets derived from five different NLL tissue types of the reference accession cv. Tanjil are described. The Tanjil transcriptome was compared to transcriptomes of an early domesticated cv. Unicrop, a wild accession P27255, as well as accession 83A:476, together being the founding parents of two recombinant inbred line (RIL) populations. In silico predictions for transcriptome-derived gene-based length and SNP polymorphic markers were conducted and corroborated using a survey assembly sequence for NLL cv. Tanjil. This yielded extensive indel and SNP polymorphic markers for the two RIL populations. A total of 335 transcriptome-derived markers and 66 BAC-end sequence-derived markers were evaluated, and 275 polymorphic markers were selected to genotype the reference NLL 83A:476 × P27255 RIL population. This significantly improved the completeness, marker density and quality of the reference NLL genetic map.

  3. Mushroom body defect, a gene involved in the control of neuroblast proliferation in Drosophila, encodes a coiled–coil protein

    Guan, Zhonghui; Prado, Antonio; Melzig, Jörg; Heisenberg, Martin; Nash, Howard A.; Raabe, Thomas


    Neurogenesis relies on the establishment of the proper number and precisely controlled proliferation of neuroblasts, the neuronal precursor cells. A role for the mushroom body defect (mud) gene in both of these aspects of neuroblast behavior, as well as possible roles in other aspects of fruit fly biology, is implied by phenotypes associated with mud mutations. We have localized mud by determining the sequence change in one point mutant, identifying a predicted ORF affected by the mutation, and showing that an appropriate segment of the genome rescues mud mutant phenotypes. An analysis of mud cDNAs and a survey of mud transcripts by Northern blotting indicate that the gene is subject to differential splicing and is expressed primarily during embryogenesis but also, at lower levels, during subsequent developmental stages in a sexually dimorphic manner. The gene is predicted to encode a polypeptide without obvious homologs but with two prominent structural features, a long coiled coil that constitutes the central core of the protein and a carboxyl-terminal transmembrane domain. PMID:10884435

  4. Effect of polymorphism in the peroxisome proliferator-activated receptor gamma gene on litter size of pigs.

    Wang, Guiying; Kong, Lujun; Hu, Peng; Fu, Jinlian; Wang, Aiguo


    The association of polymorphisms in peroxisome proliferator-activated receptor γ (PPARγ) gene with litter size was studied in Large White and Landrace pig. Three SNP loci (P1, P2 and P7) on PPARγ(2) gene were determined by PCR-SSCP and the results showed that there were A → G mutations at 220 and 324 bp in 5'-regulator region and at 147 bp in exon 6, respectively. Allele frequencies were analysed in two breeds. Information on 2341 litter records from 564 sows was used to analyse the trait total number born (TNB) and number born alive (NBA). In Large White, TNB and NBA of genotype BB for P2 locus were the lowest, and the TNB and NBA of third and following parities and all parities were 0.74 and 0.51 piglets per litter less (P NBA of the first parity of genotype BB for P1 locus were 2.0 piglets per litter higher than AA (P NBA of genotype BB were 0.66 and 0.97 piglets per litter (P NBA of the second parity of genotype AA were obviously higher than those of AB (P NBA of each parity of genotype AA were both about 2 piglets per litter more than those of BB (P < 0.05). The results indicated that PPARγ gene was significantly associated with litter size in pigs.

  5. Somatic hypermutation of immunoglobulin genes: lessons from proliferating cell nuclear antigenK164R mutant mice.

    Langerak, Petra; Krijger, Peter H L; Heideman, Marinus R; van den Berk, Paul C M; Jacobs, Heinz


    Proliferating cell nuclear antigen (PCNA) encircles DNA as a ring-shaped homotrimer and, by tethering DNA polymerases to their template, PCNA serves as a critical replication factor. In contrast to high-fidelity DNA polymerases, the activation of low-fidelity translesion synthesis (TLS) DNA polymerases seems to require damage-inducible monoubiquitylation (Ub) of PCNA at lysine residue 164 (PCNA-Ub). TLS polymerases can tolerate DNA damage, i.e. they can replicate across DNA lesions. The lack of proofreading activity, however, renders TLS highly mutagenic. The advantage is that B cells use mutagenic TLS to introduce somatic mutations in immunoglobulin (Ig) genes to generate high-affinity antibodies. Given the critical role of PCNA-Ub in activating TLS and the role of TLS in establishing somatic mutations in immunoglobulin genes, we analysed the mutation spectrum of somatically mutated immunoglobulin genes in B cells from PCNAK164R knock-in mice. A 10-fold reduction in A/T mutations is associated with a compensatory increase in G/C mutations-a phenotype similar to Poleta and mismatch repair-deficient B cells. Mismatch recognition, PCNA-Ub and Poleta probably act within one pathway to establish the majority of mutations at template A/T. Equally relevant, the G/C mutator(s) seems largely independent of PCNAK(164) modification.

  6. Genetic mapping, marker assisted selection and allelic relationships for the Pu 6 gene conferring rust resistance in sunflower.

    Bulos, Mariano; Vergani, Pablo Nicolas; Altieri, Emiliano


    Rust resistance in the sunflower line P386 is controlled by Pu 6 , a gene which was reported to segregate independently from other rust resistant genes, such as R 4 . The objectives of this work were to map Pu 6 , to provide and validate molecular tools for its identification, and to determine the linkage relationship of Pu 6 and R 4 . Genetic mapping of Pu 6 with six markers covered 24.8 cM of genetic distance on the lower end of linkage Group 13 of the sunflower consensus map. The marker most closely linked to Pu 6 was ORS316 at 2.5 cM in the distal position. ORS316 presented five alleles when was assayed with a representative set of resistant and susceptible lines. Allelism test between Pu 6 and R 4 indicated that both genes are linked at a genetic distance of 6.25 cM. This is the first confirmation based on an allelism test that at least two members of the R adv /R 4 /R 11 / R 13a /R 13b /Pu 6 cluster of genes are at different loci. A fine elucidation of the architecture of this complex locus will allow designing and constructing completely new genomic regions combining genes from different resistant sources and the elimination of the linkage drag around each resistant gene.

  7. Improved Selection with Newly Identified RAPD Markers Linked to Resistance Gene to Four Pathotypes of Colletotrichum lindemuthianum in Common Bean.

    Alzate-Marin, A L; Menarim, H; de Carvalho, G A; de Paula, T J; de Barros, E G; Moreira, M A


    ABSTRACT Three F(2) populations derived from crosses between the resistant cultivar AB 136 and the susceptible cultivar Michelite (MiA), and one F(2) population derived from a cross between AB 136 and Mexico 222 (MeA), were used to identify markers linked to anthracnose resistance genes present in cultivar AB 136. Primer OPZ04 produced a DNA band (OPZ04(560)) linked in coupling phase to the resistance gene for pathotype 89 (8.5 +/- 0.025 cM) in one population derived from the cross MiA. In the same population, primer OPZ09 produced one band (OPZ09(950)) linked in repulsion phase (20.4 +/- 0.014 cM) to the same resistance gene. The simultaneous use of markers in coupling and in repulsion phases allowed the identification of the three genotypic classes. In the other two populations from cross MiA, OPZ04(560) was linked in coupling phase to resistance genes for pathotypes 73 (2.9 +/- 0.012 cM) and 81 (2.8 +/- 0.017 cM). In population MeA, OPZ04(560) was linked in coupling phase (7.5 +/- 0.033 cM) to resistance to pathotype 64. These data suggest that a single gene or complex locus of linked resistance genes present in cultivar AB 136 confers resistance to all four pathotypes of C. lindemuthianum.

  8. BRCA1 haploinsufficiency leads to altered expression of genes involved in cellular proliferation and development.

    Harriet E Feilotter

    Full Text Available The assessment of BRCA1 and BRCA2 coding sequences to identify pathogenic mutations associated with inherited breast/ovarian cancer syndrome has provided a method to identify high-risk individuals, allowing them to seek preventative treatments and strategies. However, the current test is expensive, and cannot differentiate between pathogenic variants and those that may be benign. Focusing only on one of the two BRCA partners, we have developed a biological assay for haploinsufficiency of BRCA1. Using a series of EBV-transformed cell lines, we explored gene expression patterns in cells that were BRCA1 wildtype compared to those that carried (heterozygous BRCA1 pathogenic mutations. We identified a subset of 43 genes whose combined expression pattern is a sensitive predictor of BRCA1 status. The gene set was disproportionately made up of genes involved in cellular differentiation, lending credence to the hypothesis that single copy loss of BRCA1 function may impact differentiation, rendering cells more susceptible to undergoing malignant processes.

  9. Production and processing studies on calpain-system gene markers for beef tenderness: consumer assessments of eating quality.

    Robinson, D L; Cafe, L M; McIntyre, B L; Geesink, G H; Barendse, W; Pethick, D W; Thompson, J M; Polkinghorne, R; Greenwood, P L


    We investigated the effects of calpain-system genetic markers on consumer beef quality ratings, including interactions of marker effects with hormonal growth promotant (HGP) use and tenderstretch hanging. Brahman cattle in New South Wales (NSW; n = 164) and Western Australia (WA; n = 141) were selected at weaning from commercial and research herds to achieve balance and divergence in calpastatin (CAST) and calpain 3 (CAPN3) gene marker status. Genotypes for μ-calpain (CAPN1-4751 and CAPN1-316) were also determined. Angus cattle (49 in NSW, 17 in WA) with favorable CAST and CAPN3 alleles, balanced for CAPN1-316 status, were also studied. Half the cattle at each site had HGP (Revalor-H, containing 200 mg trenbolone acetate and 20 mg 17β-estradiol) implants during grain finishing. One side of each carcass was suspended from the Achilles tendon (AT) and the other from the pelvis [tenderstretch (TS)]. Meat Standards Australia consumer panels scored 7-d aged striploin steaks from both AT and TS sides, and 7-d aged rump and oyster blade steaks from the AT side of each carcass. Two favorable CAST alleles increased tenderness ratings of AT-striploin, TS-striploin, rump, and oyster blade steaks by, respectively, 6.1, 4.2, 4.2, and 3.1 units, and overall liking by 4.7, 2.8, 2.9, 3.7 (all P Brahman steaks from the same location with the same marker alleles had similar scores. In contrast, NSW Angus striploin steaks scored about 15 units greater for tenderness and overall liking (P < 0.001) than cattle with the same marker alleles at the other 3 location × breed combinations, which had generally similar scores. Therefore, calpain-system gene markers have beneficial effects on eating quality, consistent with our previous findings for objective meat quality.

  10. Ligands of Peroxisome Proliferator-activated Receptor Inhibit Homocysteineinduced DNA Methylation of Inducible Nitric Oxide Synthase Gene

    Yideng JIANG; Jianzhong ZHANG; Jiantuan XIONG; Jun CAO; Guizhong LI; Shuren WANG


    Homocysteine (Hcy) is a risk factor for atherosclerosis. It is generally accepted that inducible nitric oxide synthase (iNOS) is a key enzyme in the regulation of vascular disease. The aim of the present study is to investigate the effects of peroxisome proliferator-activated receptor ligands on iNOS in the presence of Hcy in human monocytes. Foam cells, induced by oxidize low density lipoprotein (ox-LDL) and phorbol myristate acetate (PMA) in the presence of different concentrations of Hcy, clofibrate and pioglitazone in human monocytes for 4 d, were examined by oil red O staining. The activity of iNOS was detected by real-time quantitative reverse transcription-polymerase chain reaction and Western blot analysis. The capability of DNA methylation was measured by assaying endogenous C5 DNA methyltransferase (C5MTase)activity, and the iNOS promoter methylation level was determined by quantitative MethyLight assays. The results indicated that Hcy increased the activity of C5MTase and the level of iNOS gene DNA methylation,resulting in a decrease of iNOS expression. Clofibrate and pioglitazone could antagonize the Hcy effect on iNOS expression through DNA methylation, resulting in attenuation of iNOS transcription. These findings suggested that Hcy decreased the expression of iNOS by elevating iNOS DNA methylation levels, which can repress the transcription of some genes. Peroxisome proliferator-activated receptor α/γ ligands can down-regulate iNOS DNA methylation, and could be useful for preventing Hcy-induced atherosclerosis by repressing iNOS expression.

  11. Development of tools and strategies towards marker assisted selection and gene cloning

    Brugmans, B.W.


    In this thesis research is described aiming at alleviation of the perceived limitations in the standard protocol which encompasses: mapping a trait, followed by marker saturation, genetic resolution, and finally BAC landing and walking to span the physical distance between the markers.

  12. Low enzymatic activity haplotypes of the human catechol-O-methyltransferase gene: enrichment for marker SNPs.

    Andrea G Nackley

    Full Text Available Catechol-O-methyltransferase (COMT is an enzyme that plays a key role in the modulation of catechol-dependent functions such as cognition, cardiovascular function, and pain processing. Three common haplotypes of the human COMT gene, divergent in two synonymous and one nonsynonymous (val(158met position, designated as low (LPS, average (APS, and high pain sensitive (HPS, are associated with experimental pain sensitivity and risk of developing chronic musculoskeletal pain conditions. APS and HPS haplotypes produce significant functional effects, coding for 3- and 20-fold reductions in COMT enzymatic activity, respectively. In the present study, we investigated whether additional minor single nucleotide polymorphisms (SNPs, accruing in 1 to 5% of the population, situated in the COMT transcript region contribute to haplotype-dependent enzymatic activity. Computer analysis of COMT ESTs showed that one synonymous minor SNP (rs769224 is linked to the APS haplotype and three minor SNPs (two synonymous: rs6267, rs740602 and one nonsynonymous: rs8192488 are linked to the HPS haplotype. Results from in silico and in vitro experiments revealed that inclusion of allelic variants of these minor SNPs in APS or HPS haplotypes did not modify COMT function at the level of mRNA folding, RNA transcription, protein translation, or enzymatic activity. These data suggest that neutral variants are carried with APS and HPS haplotypes, while the high activity LPS haplotype displays less linked variation. Thus, both minor synonymous and nonsynonymous SNPs in the coding region are markers of functional APS and HPS haplotypes rather than independent contributors to COMT activity.

  13. Identifying diagnostic endocrine markers and changes in endometrial gene expressions during pyometra in cats.

    Jursza-Piotrowska, Ewelina; Siemieniuch, Marta J


    Pyometra is a significant reproductive problem in cats. The aims of this study were to evaluate (i) the immunological profile of queens by studying plasma concentrations of metabolites of prostacyclin I2 (6-keto-PGF1α), leukotriene B4 (LTB4) and leukotriene C4 (LTC4); and (ii) the gene transcription profiles of Toll-like receptors (TLRs) 2 and 4 (TLR2/4), PGE2-synthase (PGES), PGF2α-synthase (PGFS) and prostaglandin-endoperoxide synthase 2 (PTGS2) in the feline endometrium throughout the estrous cycle, after medroxyprogesterone acetate (MPA) treatment and during pyometra. The concentration of plasma 6-keto-PGF1α in pyometra was increased in comparison to other groups studied (p<0.01). Endometrial mRNA coding for TLR2 was up-regulated in cats suffering from pyometra compared to other groups (p<0.001). Expression of mRNA for TLR4 was up-regulated in endometria originating from MPA-treated cats, pyometra and late diestrus cats, compared with tissues from cats during estrus and anestrus (p<0.05). As expected, endometrial mRNA for PTGS2 was up-regulated only in pyometra, compared with other groups (p<0.001). Similarly, endometrial mRNA for PGFS was up-regulated in pyometra, compared with endometria from anestrus, late diestrus and from MPA-treated cats (p<0.05), or from cats during estrus (p<0.01). Overall, these results indicate that plasma concentrations of LTB4 and LTC4 are not useful diagnostic markers since they were not increased in queens with pyometra, in contrast to 6-keto-PGF1α. In addition, treatment with MPA evoked neither endocrine nor molecular changes in endometria of cats.

  14. [Association between peroxisome proliferator-activated receptors gene polymorphism and essential hypertension].

    Lin, Yao; Gu, Shu-jun; Wu, Ming; Chen, Qiu; Zhou, Zheng-yuan; Yu, Hao; Zhang, Li-jun; Luo, Wen-shu; Guo, Zhi-rong


    To investigate the association between ten single nucleotide polymorphism (SNP) in the peroxisome proliferator-activated receptor (PPAR) α/δ/γ and essential hypertension (EH). Participants were recruited within the framework of a cohort populations survey from the PMMJS (Prevention of Multiple Metabolic Disorders and MS in Jiangsu Province) which was conducted in the urban community of Jiangsu province from 1999 to 2007. Eight hundred and twenty subjects (551 non-hypertensive subjects, 269 hypertensive subjects) were randomly selected but were not related to each other. Ten SNP (rs135539, rs1800206, rs4253778 of PPARα; rs2016520, rs9794 of PPARδ; rs10865710, rs1805192, rs4684847, rs709158 and rs3856806 of PPARγ) were selected from the HapMap database. χ(2) test was used to determine whether the whole population was in H-W genetic equilibrium. SHEsis software was used to examine the relations of SNP and linkage equilibrium. Logistic regression model was used to examine the association between ten SNP in the PPAR and EH. Difference on the distribution of four SNP genotypes including rs1800206, rs9794, rs10865710 and rs4684847 between high blood pressure and non-high blood pressure group, high systolic blood pressure (SBP) and normal SBP group, high diastolic blood pressure (DBP) and normal DBP group was significant (P PPARδ and rs4684847, rs10865710 of PPARγ influenced high blood pressure, high SBP and high DBP to different degrees.

  15. Molecular mapping of the hybrid necrosis gene NetJingY176 in Aegilops tauschii using microsatellite markers

    Fengbo Xue


    Full Text Available The rich genetic variation preserved in collections of Aegilops tauschii can be readily exploited to improve common wheat using synthetic hexaploid wheat lines. However, hybrid necrosis, which is characterized by progressive death of leaves or plants, has been observed in certain interspecific crosses between tetraploid wheat and Ae. tauschii. The aim of this study was to construct a fine genetic map of a gene (temporarily named NetJingY176 conferring hybrid necrosis in Ae. tauschii accession Jing Y176. A triploid F1 population derived from distant hybridization between Ae. tauschii and tetraploid wheat was used to map the gene with microsatellite markers. The newly developed markers XsdauK539 and XsdauK561 co-segregated with NetJingY176 on chromosome arm 2DS. The tightly linked markers developed in this study were used to genotype 91 Ae. tauschii accessions. The marker genotype analysis suggested that 49.45% of the Ae. tauschii accessions carry NetJingY176. Interestingly, hybrid necrosis genotypes tended to appear more commonly in Ae. tauschii ssp. tauschii than in Ae. tauschii ssp. strangulata.

  16. Molecular mapping of the hybrid necrosis gene NetJingY176 in Aegilops tauschii using microsatellite markers

    Fengbo; Xue; Jun; Guo; Changying; Guan; Hongwei; Wang; Anfei; Li; Lingrang; Kong


    The rich genetic variation preserved in collections of Aegilops tauschii can be readily exploited to improve common wheat using synthetic hexaploid wheat lines. However,hybrid necrosis, which is characterized by progressive death of leaves or plants, has been observed in certain interspecific crosses between tetraploid wheat and Ae. tauschii. The aim of this study was to construct a fine genetic map of a gene(temporarily named Net Jing Y176)conferring hybrid necrosis in Ae. tauschii accession Jing Y176. A triploid F1 population derived from distant hybridization between Ae. tauschii and tetraploid wheat was used to map the gene with microsatellite markers. The newly developed markers Xsdau K539 and Xsdau K561 co-segregated with Net Jing Y176 on chromosome arm 2DS. The tightly linked markers developed in this study were used to genotype 91 Ae. tauschii accessions. The marker genotype analysis suggested that 49.45% of the Ae. tauschii accessions carry Net Jing Y176. Interestingly, hybrid necrosis genotypes tended to appear more commonly in Ae. tauschii ssp. tauschii than in Ae. tauschii ssp. strangulata.

  17. Molecular mapping of the hybrid necrosis gene NetJingY176 in Aegilops tauschii using microsatellite markers

    Fengbo Xue; Jun Guo; Changying Guan; Hongwei Wang; Anfei Li; Lingrang Kong


    The rich genetic variation preserved in collections of Aegilops tauschii can be readily exploited to improve common wheat using synthetic hexaploid wheat lines. However, hybrid necrosis, which is characterized by progressive death of leaves or plants, has been observed in certain interspecific crosses between tetraploid wheat and Ae. tauschii. The aim of this study was to construct a fine genetic map of a gene (temporarily named NetJingY176) conferring hybrid necrosis in Ae. tauschii accession Jing Y176. A triploid F1 population derived from distant hybridization between Ae. tauschii and tetraploid wheat was used to map the gene with microsatellite markers. The newly developed markers XsdauK539 and XsdauK561 co-segregated with NetJingY176 on chromosome arm 2DS. The tightly linked markers developed in this study were used to genotype 91 Ae. tauschii accessions. The marker genotype analysis suggested that 49.45% of the Ae. tauschii accessions carry NetJingY176. Interestingly, hybrid necrosis genotypes tended to appear more commonly in Ae. tauschii ssp. tauschii than in Ae. tauschii ssp. strangulata.

  18. GLUT1 gene is a potential hypoxic marker in colorectal cancer patients

    Chang Hui-Jen


    Full Text Available Abstract Background Tumor hypoxia is an important factor related to tumor resistance to radiotherapy and chemotherapy. This study investigated molecules synthesized in colorectal cancer cells during hypoxia to explore the possibility of developing molecular probes capable of detecting cell death and/or the efficiency of radiotherapy and chemotherapy. Methods At first, we incubated two human colorectal adenocarcinoma cell lines SW480 (UICC stage II and SW620 (UICC stage III cells in hypoxic (≤2% O2, 93% N2, and 5% CO2 and normoxic conditions (20% O2, 75% N2, and 5% CO2 for 24 h and 48 h. The relative expression ratio of GLUT1 mRNA in hypoxic conditions was analyzed by RT-PCR. Ten cancerous tissues collected from human colorectal cancer patients were examined. HIF-1α and HIF-2α levels were measured to indicate the degree of hypoxia, and gene expression under hypoxic conditions was determined. As a comparison, HIF-1α, HIF-2α, and GLUT1 levels were measured in the peripheral blood of 100 CRC patients. Results Hypoxia-induced lactate was found to be elevated 3.24- to 3.36-fold in SW480 cells, and 3.06- to 3.17-fold in SW620 cells. The increased relative expression ratio of GLUT1 mRNA, under hypoxic conditions was higher in SW620 cells (1.39- to 1.72-fold elevation than in SW480 cells (1.24- to 1.66-fold elevation. HIF-1α and HIF-2α levels were elevated and GLUT1 genes were significantly overexpressed in CRC tissue specimens. The elevated ratio of GLUT1 was higher in stage III and IV CRC tissue specimens than in the stage I and II (2.97–4.73 versus 1.44–2.11. GLUT1 mRNA was also increased in the peripheral blood of stage II and III CRC patients as compared to stage I patients, suggesting that GLUT1 may serve as a hypoxic indicator in CRC patients. Conclusion In conclusion, this study demonstrated that GLUT1 has the potential to be employed as a molecular marker to indicate the degree of hypoxia experienced by tumors circulating in the

  19. Grape skin extract reduces adipogenesis- and lipogenesis-related gene expression in 3T3-L1 adipocytes through the peroxisome proliferator-activated receptor-γ signaling pathway.

    Jeong, Yoo Seok; Hong, Joo Heon; Cho, Kyung Hyun; Jung, Hee Kyoung


    We previously reported that grape skin ethanol extract (GSE) decreases adipogenic transcription factor gene expression, inhibiting triglyceride accumulation in 3T3-L1 adipocytes. In this study, we hypothesized that GSE may induce differential expression profiles in adipocytes, thus providing protection against obesity. Thirty-five genes involved in the peroxisome proliferator-activated receptor-γ (PPARγ) signaling pathway, lipid metabolism, or adipogenesis were identified through microarray analysis of adipocytes treated with GSE. Expression of the genes involved in PPARγ signaling, Adipoq, Scd1, Nr1h3, Fabp5, Scd2, and Pparg decreased with GSE treatment, whereas expression of Ppargc1a increased. Lipid metabolism-associated genes Mlxp1, Stat5a, Hsl, Plin1, and Vdr were down-regulated. Interestingly, GSE also affected expression of genes related to the mitogen-activated protein kinases pathway. GSE extract treatment decreased expression of aP2, Fas, and Tnfa, known markers of adipogenesis, as measured by real-time polymerase reaction. These findings demonstrate the antiadipogenic effects of GSE on 3T3-L1 adipocytes at the genetic level, primarily on the PPARγ signaling pathway. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  20. AFLP Marker Linked to Turnip Mosaic Virus Susceptible Gene in Chinese Cabbage (Brassica rapa L.ssp.pekinensis)

    HAN He-ping; SUN Ri-fei; ZHANG Shu-jiang; LI Fei; ZHANG Shi-fan; NIU Xin-ke


    Turnip mosaic virus (TuMV) which has several strains causes the most important virusdisease in Chinese cabbage in terms of crop damage. In China, Chinese cabbage is infectedby a mixture of strains, breeding of cultivar for the TuMV resistance has become themajor aim. Screening the molecular marker linked to the TuMV-resistance gene formolecular assisted selection is the major method to improve the breeding efficiency. Inthis study, we used AFLP technique and the method of bulked segregant analysis(BSA) tostudy the progeny of Brp0058 x Brp0108, and identified two DNA molecular marker linked toTurnip mosaic virus-resistance gene with a recombination frequency 7.5 cM and 8.4 cM.

  1. Rapid development of molecular markers by next-generation sequencing linked to a gene conferring phomopsis stem blight disease resistance for marker-assisted selection in lupin (Lupinus angustifolius L.) breeding.

    Yang, Huaan; Tao, Ye; Zheng, Zequn; Shao, Di; Li, Zhenzhong; Sweetingham, Mark W; Buirchell, Bevan J; Li, Chengdao


    Selection for phomopsis stem blight disease (PSB) resistance is one of the key objectives in lupin (Lupinus angustifolius L.) breeding programs. A cross was made between cultivar Tanjil (resistant to PSB) and Unicrop (susceptible). The progeny was advanced into F(8) recombinant inbred lines (RILs). The RIL population was phenotyped for PSB disease resistance. Twenty plants from the RIL population representing disease resistance and susceptibility was subjected to next-generation sequencing (NGS)-based restriction site-associated DNA sequencing on the NGS platform Solexa HiSeq2000, which generated 7,241 single nucleotide polymorphisms (SNPs). Thirty-three SNP markers showed the correlation between the marker genotypes and the PSB disease phenotype on the 20 representative plants, which were considered as candidate markers linked to a putative R gene for PSB resistance. Seven candidate markers were converted into sequence-specific PCR markers, which were designated as PhtjM1, PhtjM2, PhtjM3, PhtjM4, PhtjM5, PhtjM6 and PhtjM7. Linkage analysis of the disease phenotyping data and marker genotyping data on a F(8) population containing 187 RILs confirmed that all the seven converted markers were associated with the putative R gene within the genetic distance of 2.1 CentiMorgan (cM). One of the PCR markers, PhtjM3, co-segregated with the R gene. The seven established PCR markers were tested in the 26 historical and current commercial cultivars released in Australia. The numbers of "false positives" (showing the resistance marker allele band but lack of the putative R gene) for each of the seven PCR markers ranged from nil to eight. Markers PhtjM4 and PhtjM7 are recommended in marker-assisted selection for PSB resistance in the Australian national lupin breeding program due to its wide applicability on breeding germplasm and close linkage to the putative R gene. The results demonstrated that application of NGS technology is a rapid and cost-effective approach in

  2. De novo assembly, gene annotation and marker development using Illumina paired-end transcriptome sequences in celery (Apium graveolens L..

    Nan Fu

    Full Text Available BACKGROUND: Celery is an increasing popular vegetable species, but limited transcriptome and genomic data hinder the research to it. In addition, a lack of celery molecular markers limits the process of molecular genetic breeding. High-throughput transcriptome sequencing is an efficient method to generate a large transcriptome sequence dataset for gene discovery, molecular marker development and marker-assisted selection breeding. PRINCIPAL FINDINGS: Celery transcriptomes from four tissues were sequenced using Illumina paired-end sequencing technology. De novo assembling was performed to generate a collection of 42,280 unigenes (average length of 502.6 bp that represent the first transcriptome of the species. 78.43% and 48.93% of the unigenes had significant similarity with proteins in the National Center for Biotechnology Information (NCBI non-redundant protein database (Nr and Swiss-Prot database respectively, and 10,473 (24.77% unigenes were assigned to Clusters of Orthologous Groups (COG. 21,126 (49.97% unigenes harboring Interpro domains were annotated, in which 15,409 (36.45% were assigned to Gene Ontology(GO categories. Additionally, 7,478 unigenes were mapped onto 228 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG. Large numbers of simple sequence repeats (SSRs were indentified, and then the rate of successful amplication and polymorphism were investigated among 31 celery accessions. CONCLUSIONS: This study demonstrates the feasibility of generating a large scale of sequence information by Illumina paired-end sequencing and efficient assembling. Our results provide a valuable resource for celery research. The developed molecular markers are the foundation of further genetic linkage analysis and gene localization, and they will be essential to accelerate the process of breeding.

  3. Identification of a RAPD marker linked to the Co-6 anthracnose resistant gene in common bean cultivar AB 136

    Alzate-Marin Ana Lilia


    Full Text Available The pathogenic variability of the fungus Colletotrichum lindemuthianum represents an obstacle for the creation of resistant common bean (Phaseolus vulgaris L. varieties. Gene pyramiding is an alternative strategy for the development of varieties with durable resistance. RAPD markers have been proposed as a means to facilitate pyramiding of resistance genes without the need for multiple inoculations of the pathogens. The main aims of this work were to define the inheritance pattern of resistance present in common bean cultivar AB 136 in segregating populations derived from crosses with cultivar Rudá (susceptible to most C. lindemuthianum races and to identify RAPD markers linked to anthracnose resistance. The two progenitors, populations F1 and F2, F2:3 families and backcross-derived plants were inoculated with race 89 of C. lindemuthianum under environmentally controlled greenhouse conditions. The results indicate that a single dominant gene, Co-6, controls common bean resistance to this race, giving a segregation ratio between resistant and susceptible plants of 3:1 in the F2, 1:0 in the backcrosses to AB 136 and 1:1 in the backcross to Rudá. The segregation ratio of F2:3 families derived from F2 resistant plants was 1:2 (homozygous to heterozygous resistant. Molecular marker analyses in the F2 population identified a DNA band of approximately 940 base pairs (OPAZ20(940, linked in coupling phase at 7.1 cM of the Co-6 gene. This marker is being used in our backcross breeding program to develop Rudá-derived common bean cultivars resistant to anthracnose and adapted to central Brazil.

  4. A conservative region of the mercuric reductase gene (mera) as a molecular marker of bacterial mercury resistance

    Sotero-Martins, Adriana; de Jesus, Michele Silva; Lacerda, Michele; Moreira, Josino Costa; Filgueiras, Ana Luzia Lauria; Barrocas, Paulo Rubens Guimarães


    The most common bacterial mercury resistance mechanism is based on the reduction of Hg(II) to Hg0, which is dependent of the mercuric reductase enzyme (MerA) activity. The use of a 431 bp fragment of a conservative region of the mercuric reductase (merA) gene was applied as a molecular marker of this mechanism, allowing the identification of mercury resistant bacterial strains. PMID:24031221

  5. Integration of gene-based markers in a pearl millet genetic map for identification of candidate genes underlying drought tolerance quantitative trait loci

    Sehgal Deepmala


    Full Text Available Abstract Background Identification of genes underlying drought tolerance (DT quantitative trait loci (QTLs will facilitate understanding of molecular mechanisms of drought tolerance, and also will accelerate genetic improvement of pearl millet through marker-assisted selection. We report a map based on genes with assigned functional roles in plant adaptation to drought and other abiotic stresses and demonstrate its use in identifying candidate genes underlying a major DT-QTL. Results Seventy five single nucleotide polymorphism (SNP and conserved intron spanning primer (CISP markers were developed from available expressed sequence tags (ESTs using four genotypes, H 77/833-2, PRLT 2/89-33, ICMR 01029 and ICMR 01004, representing parents of two mapping populations. A total of 228 SNPs were obtained from 30.5 kb sequenced region resulting in a SNP frequency of 1/134 bp. The positions of major pearl millet linkage group (LG 2 DT-QTLs (reported from crosses H 77/833-2 × PRLT 2/89-33 and 841B × 863B were added to the present consensus function map which identified 18 genes, coding for PSI reaction center subunit III, PHYC, actin, alanine glyoxylate aminotransferase, uridylate kinase, acyl-CoA oxidase, dipeptidyl peptidase IV, MADS-box, serine/threonine protein kinase, ubiquitin conjugating enzyme, zinc finger C- × 8-C × 5-C × 3-H type, Hd3, acetyl CoA carboxylase, chlorophyll a/b binding protein, photolyase, protein phosphatase1 regulatory subunit SDS22 and two hypothetical proteins, co-mapping in this DT-QTL interval. Many of these candidate genes were found to have significant association with QTLs of grain yield, flowering time and leaf rolling under drought stress conditions. Conclusions We have exploited available pearl millet EST sequences to generate a mapped resource of seventy five new gene-based markers for pearl millet and demonstrated its use in identifying candidate genes underlying a major DT-QTL in this species. The reported gene

  6. Influence of Ginkgo biloba extract on the proliferation, apoptosis of ACC-2 cell and Survivin gene expression in adenoid cystic carcinoma of lacrimal gland

    Li-Xiao Zhou; Yu Zhu


    Objective: To explore the influence of extract of Ginkgo biloba (EGB) on the proliferation, apoptosis of ACC-2 cell and Survivin gene expression in adenoid cystic carcinoma (ACC) of lacrimal gland. Methods:ACC-2 cell in human with ACC of lacrimal gland was in vitro cultured. MTT method was used for cell proliferation detection. Annexin V/PI double-staining flow cytometer was used to detect cell apoptosis and cell cycle. Survivin gene expression was analyzed by RT-PCR and Western blotting. Results: EGB had inhibitory effect on the proliferation of ACC-2 cell with significant dose-effect relationship, and there was statistical difference when compared with the control group (P<0.01). The inhibitory concentration 50 % (IC50) is 88 mg/L. The flow cytometer test indicated that EGB can gradually increase ACC-2 cell in G0-G1 stage and decrease it in G2-M and S stage. With the increase of dose, the apoptosis rate of ACC-2 cell was obviously increased (P<0.05 or P<0.01). EGB had certain inhibitory effect on Survivin gene expression of ACC-2 cell, and Survivin gene expression was decreased with the increasing of the EGB concentration (P<0.01). Conclusions:EGB can effectively inhibit Survivin gene expression of ACC-2 cell in human with ACC of lacrimal gland, induce the apoptosis of ACC-2 cell and inhibit tumor cell proliferation.

  7. The labial gene is required to terminate proliferation of identified neuroblasts in postembryonic development of the Drosophila brain

    Philipp A. Kuert


    The developing brain of Drosophila has become a useful model for studying the molecular genetic mechanisms that give rise to the complex neuronal arrays that characterize higher brains in other animals including mammals. Brain development in Drosophila begins during embryogenesis and continues during a subsequent postembryonic phase. During embryogenesis, the Hox gene labial is expressed in the developing tritocerebrum, and labial loss-of-function has been shown to be associated with a loss of regional neuronal identity and severe patterning defects in this part of the brain. However, nothing is known about the expression and function of labial, or any other Hox gene, during the postembryonic phase of brain development, when the majority of the neurons in the adult brain are generated. Here we report the first analysis of Hox gene action during postembryonic brain development in Drosophila. We show that labial is expressed initially in six larval brain neuroblasts, of which only four give rise to the labial expressing neuroblast lineages present in the late larval brain. Although MARCM-based clonal mutation of labial in these four neuroblast lineages does not result in an obvious phenotype, a striking and unexpected effect of clonal labial loss-of-function does occur during postembryonic brain development, namely the formation of two ectopic neuroblast lineages that are not present in wildtype brains. The same two ectopic neuroblast lineages are also observed following cell death blockage and, significantly, in this case the resulting ectopic lineages are Labial-positive. These findings imply that labial is required in two specific neuroblast lineages of the wildtype brain for the appropriate termination of proliferation through programmed cell death. Our analysis of labial function reveals a novel cell autonomous role of this Hox gene in shaping the lineage architecture of the brain during postembryonic development.

  8. Inheritance of Resistance to SMV3 and Identification of RAPD Marker Linked to the Resistant Gene in Soybean

    ZHENG Cui-ming; CHANG Ru-zhen; QIU Li-juan


    One SMV resistant soybean line (95-5383) was crossed with four susceptible soybean varieties/line ( HB1, Tiefeng21, Amsoy, Williams) and one resistant introduced line PI486355. Their F1 and F2individuals were identified for SMV resistance by inoculation with SMV3. The results showed that in the four crosses of resistant × susceptible, F1 were susceptible and the ratio of F2 populations was 1 resistant : 3susceptible (mosaic and necrosis), indicating that 95-5383 carries one recessive gene that confer resistance to SMV3. There is segregation of susceptibility in F2 progenies from the cross of 95-5383 × PI486355, indicating that the SMV3 resistant gene in 95-5383 is located at different locus from PI486355. By bulked segregating analysis (BSA) in F2 populations of 95-5383 × HB1, one codominant RAPD marker OPN11980/1070 closely linked to SMV3 resistance gene amplified with RAPD primer OPN11 was identified. The DNA fragment OPN11980 was amplified in resistant parent 95-5383 and resistant bulk, and OPN111070 was amplified in susceptible parent HB1 and susceptible bulk. OPN11980/1070 was amplified in F1. Identification of the markers in F2 plants showed that the codominant marker OPN11980/1070 is closely linked to the SMV resistance locus in95-5383, with genetic distance of 2.1cM.

  9. Marker-assisted introgression of broad-spectrum blast resistance genes into the cultivated MR219 rice variety.

    Miah, Gous; Rafii, Mohd Y; Ismail, Mohd R; Puteh, Adam B; Rahim, Harun A; Latif, Mohammad A


    The rice cultivar MR219 is famous for its better yield and long and fine grain quality; however, it is susceptible to blast disease. The main objective of this study was to introgress blast resistance genes into MR219 through marker-assisted selection (MAS). The rice cultivar MR219 was used as the recurrent parent, and Pongsu Seribu 1 was used as the donor. Marker-assisted foreground selection was performed using RM6836 and RM8225 to identify plants possessing blast resistance genes. Seventy microsatellite markers were used to estimate recurrent parent genome (RPG) recovery. Our analysis led to the development of 13 improved blast resistant lines with Piz, Pi2 and Pi9 broad-spectrum blast resistance genes and an MR219 genetic background. The RPG recovery of the selected improved lines was up to 97.70% with an average value of 95.98%. Selected improved lines showed a resistance response against the most virulent blast pathogen pathotype, P7.2. The selected improved lines did not express any negative effect on agronomic traits in comparison with MR219. The research findings of this study will be a conducive approach for the application of different molecular techniques that may result in accelerating the development of new disease-resistant rice varieties, which in turn will match rising demand and food security worldwide. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  10. Polymorphisms of the peroxisome proliferator-activated receptor γ (PPARγ) gene are associated with osteoporosis

    Harsløf, T; Tofteng, C L; Husted, L B


    Stimulation of PPARγ turns mesenchymal stem cells into adipocytes instead of osteoblasts. We investigated the effect of polymorphisms in the PPARγ gene on BMD and fracture risk in two Danish cohorts and found opposing effects of certain SNPs and haplotypes in the two cohorts probably owing...... to environmental factors. INTRODUCTION: Stimulation of PPARγ causes development of mesenchymal stem cells to adipocytes instead of osteoblasts leading to decreased osteoblast number and BMD. The aim of this study was to examine the effect of PPARG polymorphisms on BMD and fracture risk in two Danish cohorts: AROS...

  11. Development of a PCR marker for rapid identification of the Bt-10 gene for common bunt resistance in wheat.

    Laroche, A; Demeke, T; Gaudet, D A; Puchalski, B; Frick, M; McKenzie, R


    In western Canada, the Bt-10 resistance gene in wheat (Triticum aestivum) is effective against all the known races of common bunt caused by Tilletia tritici and T laevis. The genotypes of 199 F2 plants, originated from a cross between BW553 containing Bt-10 and the susceptible spring wheat cultivar 'Neepawa,' were established in greenhouse and field inoculation studies. A ratio of 1:2:1 resistant : heterozygous : susceptible was observed for bunt reaction, indicating that Bt-10 was expressed in a partially dominant fashion. A polymorphic DNA fragment, amplified using RAPD, and previously shown to be linked to Bt-10 was sequenced and SCAR (sequence characterized amplified region) primers devised. However, SCAR primers failed to amplify the polymorphic fragment. Restriction of PCR products with DraI revealed a polymorphic fragment of 490 bp resulting from a single base pair difference between lines possessing Bt-10 and those lacking the gene. As per the base pair difference, FSD and RSA primers were designed to generate a 275-bp polymorphic DNA fragment. Both 275- and 490-bp polymorphic fragments were present in all of the 22 cultivars known to carry Bt-10, and absent in all 16 cultivars lacking Bt-10. A 3:1 ratio was observed for presence: absence of the 275-bp marker in the F2 population. Using Southern analysis, the 490-bp fragment was effective in differentiating homozygous resistant plants from those heterozygous for Bt-10, based on its presence and the hybridization signal strength. A 1:2:1 resistant : heterozygous : susceptible ratio was also observed for the molecular marker and corresponded to 88% of the phenotypes deduced from the original F2 population. The molecular marker was estimated to be between 1.1 cM and 6.5 cM away from the Bt-10 resistance gene, based on the segregation analysis. Segregation analyses of Bt-10 and the 275-bp marker, evaluated in three different Canada Prairie Spring (CPS) wheat populations, demonstrated a segregation ratio of 3

  12. aes, the gene encoding the esterase B in Escherichia coli, is a powerful phylogenetic marker of the species

    Tuffery Pierre


    Full Text Available Abstract Background Previous studies have established a correlation between electrophoretic polymorphism of esterase B, and virulence and phylogeny of Escherichia coli. Strains belonging to the phylogenetic group B2 are more frequently implicated in extraintestinal infections and include esterase B2 variants, whereas phylogenetic groups A, B1 and D contain less virulent strains and include esterase B1 variants. We investigated esterase B as a marker of phylogeny and/or virulence, in a thorough analysis of the esterase B-encoding gene. Results We identified the gene encoding esterase B as the acetyl-esterase gene (aes using gene disruption. The analysis of aes nucleotide sequences in a panel of 78 reference strains, including the E. coli reference (ECOR strains, demonstrated that the gene is under purifying selection. The phylogenetic tree reconstructed from aes sequences showed a strong correlation with the species phylogenetic history, based on multi-locus sequence typing using six housekeeping genes. The unambiguous distinction between variants B1 and B2 by electrophoresis was consistent with Aes amino-acid sequence analysis and protein modelling, which showed that substituted amino acids in the two esterase B variants occurred mostly at different sites on the protein surface. Studies in an experimental mouse model of septicaemia using mutant strains did not reveal a direct link between aes and extraintestinal virulence. Moreover, we did not find any genes in the chromosomal region of aes to be associated with virulence. Conclusion Our findings suggest that aes does not play a direct role in the virulence of E. coli extraintestinal infection. However, this gene acts as a powerful marker of phylogeny, illustrating the extensive divergence of B2 phylogenetic group strains from the rest of the species.

  13. Homeobox gene expression profile indicates HOXA5 as a candidate prognostic marker in oral squamous cell carcinoma.

    Rodini, Camila Oliveira; Xavier, Flávia Caló Aquino; Paiva, Katiúcia Batista Silva; De Souza Setúbal Destro, Maria Fernanda; Moyses, Raquel Ajub; Michaluarte, Pedro; Carvalho, Marcos Brasilino; Fukuyama, Erica Erina; Tajara, Eloiza Helena; Okamoto, Oswaldo Keith; Nunes, Fabio Daumas


    The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC.

  14. Phylogenetic study of Geitlerinema and Microcystis (Cyanobacteria) using PC-IGS and 16S-23S ITS as markers: investigation of horizontal gene transfer.

    Piccin-Santos, Viviane; Brandão, Marcelo Mendes; Bittencourt-Oliveira, Maria Do Carmo


    Selection of genes that have not been horizontally transferred for prokaryote phylogenetic inferences is regarded as a challenging task. The markers internal transcribed spacer of ribosomal genes (16S-23S ITS) and phycocyanin intergenic spacer (PC-IGS), based on the operons of ribosomal and phycocyanin genes respectively, are among the most used markers in cyanobacteria. The region of the ribosomal genes has been considered stable, whereas the phycocyanin operon may have undergone horizontal transfer. To investigate the occurrence of horizontal transfer of PC-IGS, phylogenetic trees of Geitlerinema and Microcystis strains were generated using PC-IGS and 16S-23S ITS and compared. Phylogenetic trees based on the two markers were mostly congruent for Geitlerinema and Microcystis, indicating a common evolutionary history among ribosomal and phycocyanin genes with no evidence for horizontal transfer of PC-IGS. Thus, PC-IGS is a suitable marker, along with 16S-23S ITS for phylogenetic studies of cyanobacteria.

  15. Knockdown of astrocyte elevated gene-1 inhibits proliferation and enhancing chemo-sensitivity to cisplatin or doxorubicin in neuroblastoma cells

    Xie Li


    Full Text Available Abstract Background Astrocyte elevated gene-1 (AEG-1 was originally characterized as a HIV-1-inducible gene in primary human fetal astrocyte. Recent studies highlight a potential role of AEG-1 in promoting tumor progression and metastasis. The aim of this study was to investigate if AEG-1 serves as a potential therapeutic target of human neuroblastoma. Methods We employed RNA interference to reduce AEG-1 expression in human neuroblastoma cell lines and analyzed their phenotypic changes. Results We found that the knockdown of AEG-1 expression in human neuroblastoma cells significantly inhibited cell proliferation and apoptosis. The specific downregulation induced cell arrest in the G0/G1 phase of cell cycle. In the present study, we also observed a significant enhancement of chemo-sensitivity to cisplatin and doxorubicin by knockdown of AEG-1. Conclusion Our study suggests that overexpressed AEG-1 enhance the tumorogenic properties of neuroblastoma cells. The inhibition of AEG-1 expression could be a new adjuvant therapy for neuroblastoma.

  16. Association between the Pro12Ala Polymorphism of the Peroxisome Proliferator-Activated Receptor Gamma Gene and Strength Athlete Status

    Maciejewska-Karlowska, Agnieszka; Sawczuk, Marek; Cieszczyk, Pawel; Zarebska, Aleksandra; Sawczyn, Stanislaw


    Background The 12Ala allele of the Peroxisome Proliferator-Activated Receptor gamma gene (PPARG) Pro12Ala polymorphism produces a decreased binding affinity of the PPARγ2 protein, resulting in low activation of the target genes. The 12Ala allele carriers display a significantly improved insulin sensitivity that may result in better glucose utilisation in working skeletal muscles. We hypothesise that the PPARG 12Ala allele could be associated with strength athlete status in Polish athletes. Methodology The genotype distribution of PPARG Pro12Ala was examined in 660 Polish athletes. The athletes were stratified into four subgroups: endurance, strength-endurance, sprint-strength and strength. Control samples were prepared from 684 unrelated sedentary volunteers. A χ2 test was used to compare the PPARG Pro12Ala allele and genotype frequencies between the different groups of athletes and control subjects. Bonferroni’s correction for multiple testing was applied. Results A statistically significant higher frequency of PPARG 12Ala alleles was observed in the subgroup of strength athletes performing short-term and very intense exertion characterised by predominant anaerobic energy production (13.2% vs. 7.5% in controls; P = 0.0007). Conclusion The PPARG 12Ala allele may be a relevant genetic factor favouring strength abilities in professional athletes, especially in terms of insulin-dependent metabolism, a shift of the energy balance towards glucose utilisation and the development of a favourable weight-to-strength ratio. PMID:23799144

  17. Association between the Pro12Ala polymorphism of the peroxisome proliferator-activated receptor gamma gene and strength athlete status.

    Maciejewska-Karlowska, Agnieszka; Sawczuk, Marek; Cieszczyk, Pawel; Zarebska, Aleksandra; Sawczyn, Stanislaw


    The 12Ala allele of the Peroxisome Proliferator-Activated Receptor gamma gene (PPARG) Pro12Ala polymorphism produces a decreased binding affinity of the PPARγ2 protein, resulting in low activation of the target genes. The 12Ala allele carriers display a significantly improved insulin sensitivity that may result in better glucose utilisation in working skeletal muscles. We hypothesise that the PPARG 12Ala allele could be associated with strength athlete status in Polish athletes. The genotype distribution of PPARG Pro12Ala was examined in 660 Polish athletes. The athletes were stratified into four subgroups: endurance, strength-endurance, sprint-strength and strength. Control samples were prepared from 684 unrelated sedentary volunteers. A χ(2) test was used to compare the PPARG Pro12Ala allele and genotype frequencies between the different groups of athletes and control subjects. Bonferroni's correction for multiple testing was applied. A statistically significant higher frequency of PPARG 12Ala alleles was observed in the subgroup of strength athletes performing short-term and very intense exertion characterised by predominant anaerobic energy production (13.2% vs. 7.5% in controls; P = 0.0007). The PPARG 12Ala allele may be a relevant genetic factor favouring strength abilities in professional athletes, especially in terms of insulin-dependent metabolism, a shift of the energy balance towards glucose utilisation and the development of a favourable weight-to-strength ratio.

  18. Association between the Pro12Ala polymorphism of the peroxisome proliferator-activated receptor gamma gene and strength athlete status.

    Agnieszka Maciejewska-Karlowska

    Full Text Available BACKGROUND: The 12Ala allele of the Peroxisome Proliferator-Activated Receptor gamma gene (PPARG Pro12Ala polymorphism produces a decreased binding affinity of the PPARγ2 protein, resulting in low activation of the target genes. The 12Ala allele carriers display a significantly improved insulin sensitivity that may result in better glucose utilisation in working skeletal muscles. We hypothesise that the PPARG 12Ala allele could be associated with strength athlete status in Polish athletes. METHODOLOGY: The genotype distribution of PPARG Pro12Ala was examined in 660 Polish athletes. The athletes were stratified into four subgroups: endurance, strength-endurance, sprint-strength and strength. Control samples were prepared from 684 unrelated sedentary volunteers. A χ(2 test was used to compare the PPARG Pro12Ala allele and genotype frequencies between the different groups of athletes and control subjects. Bonferroni's correction for multiple testing was applied. RESULTS: A statistically significant higher frequency of PPARG 12Ala alleles was observed in the subgroup of strength athletes performing short-term and very intense exertion characterised by predominant anaerobic energy production (13.2% vs. 7.5% in controls; P = 0.0007. CONCLUSION: The PPARG 12Ala allele may be a relevant genetic factor favouring strength abilities in professional athletes, especially in terms of insulin-dependent metabolism, a shift of the energy balance towards glucose utilisation and the development of a favourable weight-to-strength ratio.

  19. Inhibition of periostin gene expression via RNA interference suppressed the proliferation, apoptosis and invasion in U2OS cells

    LIU Chang; HUANG Si-jian; QIN Ze-lian


    Background Periostin originally designated osteoblast-specific factor 2 (OSF-2) is frequently found to be highly expressed in various types of human cancer cell lines in vitro and human cancer tissues in vivo. We proposed that periostin was a key factor during the process of proliferation and invasion in cancer cells. We investigated the effect of periostin on the function of human osteosarcoma cell line (U2OS), such as proliferation, apoptosis, invasion and the associated signal pathway.Methods A human PGCsi/U6 promoter-driven DNA template was adopted to induce short hairpin RNA (shRNA)-triggered RNA interference (RNAi) to block periostin gene expression in the cell line U2OS. U2OS cells were divided into three groups: cells transfected with phosphate buffered saline as control group (the U2OS group), cells transfected with pGCsi as negative control group (the NC group) and cells transfected with periostin/pGCsi as experimental group (the pGCsi-periostin group). Then, transfection efficiency of cell was observed under fluorescent microscope. The expressions of periostin and the related genes in cells were detected by reverse transcription polymerase chain reaction and Western Blotting. Cell viability was determined using the methyl-thiazolyl tetrazolium bromide (MTT) quantitative colorimetric assay. The invasion and migration capability of cells were tested by transwell plates with or without extracellular matrix gel. Furthermore, the changes of cell cycle and apoptosis were analyzed by flow cytometry.Results The transfection efficiency of periostin/pGCsi to U2OS cells was about 70%-80%. When compared with the NC group, the levels of mRNA and protein of periostin in the pGCsi-periostin group decreased by 82% (F=564.71, P<0.001) and 58% (F=341.51, P <0.001 ), respectively. Meantime, the earlier apoptosis value increased by 417 (F=28.69,P <0.001). The percentage of S phase pGCsi-periostin cells decreased by 21% (F=47.00, P <0.001), however, that of G0-G1

  20. Heat shock induced excision of selectable marker genes in transgenic banana by the Cre-lox site-specific recombination system.

    Chong-Pérez, Borys; Kosky, Rafael G; Reyes, Maritza; Rojas, Luis; Ocaña, Bárbara; Tejeda, Marisol; Pérez, Blanca; Angenon, Geert


    Selectable marker genes are indispensable for efficient production of transgenic events, but are no longer needed after the selection process and may cause public concern and technological problems. Although several gene excision systems exist, few have been optimized for vegetatively propagated crops. Using a Cre-loxP auto-excision strategy, we obtained transgenic banana plants cv. Grande Naine (Musa AAA) devoid of the marker gene used for selection. We used T-DNA vectors with the cre recombinase gene under control of a heat shock promoter and selectable marker gene cassettes placed between two loxP sites in direct orientation, and a gene of interest inserted outside of the loxP sites. Heat shock promoters pGmHSP17.6-L and pHSP18.2, from soybean and Arabidopsis respectively, were tested. A transient heat shock treatment of primary transgenic embryos was sufficient for inducing cre and excising cre and the marker genes. Excision efficiency, as determined by PCR and Southern hybridization was 59.7 and 40.0% for the GmHSP17.6-L and HSP18.2 promoters, respectively. Spontaneous excision was not observed in 50 plants derived from untreated transgenic embryos. To our knowledge this is the first report describing an efficient marker gene removal system for banana. The method described is simple and might be generally applicable for the production of marker-free transgenic plants of many crop species.