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At least 865 records · Page 48Linked to original sources
PubMed1997

Use of transgenic and gene-targeted mice to model the genetic basis of hypertensive disorders.

As both essential hypertension and hypertension associated with pregnancy (pre-eclampsia) have been determined to have strong genetic components, considerable recent research has focused on identifying genes that may predispose to the development of these disorders. Recent advances in molecular genetics and the work of the Human Genome Project have facilitated the identification of genes that may be linked to these hypertensive disorders. Although molecular genetic studies performed in humans and animals can be used to link genes or mutations in genes to hypertension (once identified), studies are needed to assess their biochemical and physiologic importance. In this review, we discuss the ever-increasing importance and use of transgenic and gene-targeted mice in modeling the genetic basis of hypertensive disorders.

Animals↗
PubMed2001

Scalloped and strawberry notch are target genes of Notch signaling in the context of wing margin formation in Drosophila.

The Notch pathway regulates the differentiation of many cell types throughout development of higher metazoa. Different cellular responses are elicited through specific activation of distinct Notch target genes. In the Drosophila wing, for example, the cut gene is activated by Notch signaling along the dorso-ventral boundary but, as we show here, not in other cell types. We identify additional regulatory components, scalloped and strawberry notch, that are targets of the Notch pathway specifically within the wing anlagen. As suggested by physical interactions, these proteins could be co-factors of the cut trans-regulator Vestigial. Additional regulatory input comes from the Wingless pathway. Our data support a model, whereby context specific involvement of distinct co-regulators modulates Notch target gene activation.

Alleles↗
PubMed2006

MYC-containing double minutes in hematologic malignancies: evidence in favor of the episome model and exclusion of MYC as the target gene.

Double minutes (dmin)-circular, extra-chromosomal amplifications of specific acentric DNA fragments-are relatively frequent in malignant disorders, particularly in solid tumors. In acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), dmin are observed in approximately 1% of the cases. Most of them consist of an amplified segment from chromosome band 8q24, always including the MYC gene. Besides this information, little is known about their internal structure. We have characterized in detail the genomic organization of 32 AML and two MDS cases with MYC-containing dmin. The minimally amplified region was shown to be 4.26 Mb in size, harboring five known genes, with the proximal and the distal amplicon breakpoints clustering in two regions of approximately 500 and 600 kb, respectively. Interestingly, in 23 (68%) of the studied cases, the amplified region was deleted in one of the chromosome 8 homologs at 8q24, suggesting excision of a DNA segment from the original chromosomal location according to the 'episome model'. In one case, sequencing of both the dmin and del(8q) junctions was achieved and provided definitive evidence in favor of the episome model for the formation of dmin. Expression status of the TRIB1 and MYC genes, encompassed by the minimally amplified region, was assessed by northern blot analysis. The TRIB1 gene was found over-expressed in only a subset of the AML/MDS cases, whereas MYC, contrary to expectations, was always silent. The present study, therefore, strongly suggests that MYC is not the target gene of the 8q24 amplifications.

Adult↗
PubMed2002

Identification of direct p73 target genes combining DNA microarray and chromatin immunoprecipitation analyses.

The newly discovered p53 family member, p73, has a striking homology to p53 in both sequence and modular structure. Ectopic expression of p73 promotes transcription of p53 target genes and recapitulates the most characterized p53 biological effects such as growth arrest, apoptosis, and differentiation. Unlike p53-deficient mice that develop normally but are subject to spontaneous tumor formation, p73-deficient mice exhibit severe defects in the development of central nervous system and suffer from inflammation but are not prone to tumor development. These phenotypes suggest different biological activities mediated by p53 and p73 that might reflect activation of specific sets of target genes. Here, we have analyzed the gene expression profile of H1299 cells after p73alpha or p53 activation using oligonucleotide microarrays capable of detecting approximately 11,000 mRNA species. Our results indicate that p73alpha and p53 activate both common and distinct groups of genes. We found 141 and 320 genes whose expression is modulated by p73alpha and p53, respectively. p73alpha up-regulates 85 genes, whereas p53 induces 153 genes, of which 27 are in common with p73alpha. Functional classification of these genes reveals that they are involved in many aspects of cell function ranging from cell cycle and apoptosis to DNA repair. Furthermore, we report that some of the up-regulated genes are directly activated by p73alpha or p53.

Base Sequence↗
PubMed2001

Novel NEMO/IkappaB kinase and NF-kappa B target genes at the pre-B to immature B cell transition.

The IkappaB kinase (IKK) signaling complex is responsible for activating NF-kappaB-dependent gene expression programs. Even though NF-kappaB-responsive genes are known to orchestrate stress-like responses, critical gaps in our knowledge remain about the global effects of NF-kappaB activation on cellular physiology. DNA microarrays were used to compare gene expression programs in a model system of 70Z/3 murine pre-B cells versus their IKK signaling-defective 1.3E2 variant with lipopolysaccharide (LPS), interleukin-1 (IL-1), or a combination of LPS + phorbol 12-myristate 13-acetate under brief (2 h) or long term (12 h) stimulation. 70Z/3-1.3E2 cells lack expression of NEMO/IKKgamma/IKKAP-1/FIP-3, an essential positive effector of the IKK complex. Some stimulated hits were known NF-kappaB target genes, but remarkably, the vast majority of the up-modulated genes and an unexpected class of repressed genes were all novel targets of this signaling pathway, encoding transcription factors, receptors, extracellular ligands, and intracellular signaling factors. Thirteen stimulated (B-ATF, Pim-2, MyD118, Pea-15/MAT1, CD82, CD40L, Wnt10a, Notch 1, R-ras, Rgs-16, PAC-1, ISG15, and CD36) and five repressed (CCR2, VpreB, lambda5, SLPI, and CMAP/Cystatin7) genes, respectively, were bona fide NF-kappaB targets by virtue of their response to a transdominant IkappaBalphaSR (super repressor). MyD118 and ISG15, although directly induced by LPS stimulation, were unaffected by IL-1, revealing the existence of direct NF-kappaB target genes, which are not co-induced by the LPS and IL-1 Toll-like receptors.

Animals↗
PubMed1992

Assaying synthetic ribozymes in plants: high-level expression of a functional hammerhead structure fails to inhibit target gene activity in transiently transformed protoplasts.

A hammerhead ribozyme designed against the mRNA coding for the Escherichia coli beta-glucuronidase (GUS) reporter enzyme was constructed. The synthetic ribozyme appeared able to correctly cleave in vitro the target RNA. This catalytic molecule was then assayed for in vivo activity in plant protoplasts. Plasmids coding either for the ribozyme or for the GUS target gene were cotransfected into the cells by the PEG-calcium procedure and GUS gene expression monitored following transient expression by measuring the intracellular GUS enzymatic activity. Expression of the ribozyme to high molar excess over the GUS transcript did not lead to any significant decrease of GUS activity in the transfected protoplasts. Insertion of the ribozyme sequence in the 3'-untranslated region of the GUS mRNA also had no detectable effect on GUS reporter gene expression whereas the corresponding RNA appeared able to self-cleave in vitro. These results indicate that the ability of ribozymes to perform catalytic cleavage of their substrate mRNA in vitro is essential but clearly not sufficient to ensure that efficient inhibition of the corresponding target gene will occur upon endogenous expression of this catalytic RNA in the plant cell.

Arabidopsis↗
PubMed2004

Identification of aryl hydrocarbon receptor as a putative Wnt/beta-catenin pathway target gene in prostate cancer cells.

Recent genetic and functional analyses have implicated the wnt/beta-catenin signaling pathway in prostate cancer (CaP) pathogenesis. Thus, there is much interest in understanding the consequences of wnt signaling in CaP; target gene expression is one important area of inquiry and is the focus of this report. Adenoviral-mediated overexpression of a mutant, hyperactive form of beta-catenin in CWR22-Rv1 CaP cells led to increased aryl hydrocarbon receptor (AhR, or dioxin receptor) and transmembrane protein 2 RNA transcript expression, as detected by cDNA-microarray analyses. Validating these results, reverse transcription-PCR assays demonstrated that in CWR22-Rv1 cells as well as in LAPC-4 CaP cells, increased putative target gene RNA expression occurs with transient overexpression of mutant beta-catenin, treatment of cells with lithium chloride, or with wnt3a-conditioned medium, three distinct modes of experimental wnt/beta-catenin pathway activation. This beta-catenin-associated expression of AhR and transmembrane protein 2 does not require de novo protein synthesis and may only involve a certain subset of CaP cell lines. Western and immunofluorescence analyses were undertaken to assess the relationship between the wnt/beta-catenin-stimulated increase in AhR transcripts and AhR protein expression; we provide evidence that an association exists whereby up-regulation of AhR RNA by wnt or beta-catenin is coupled with augmented AhR protein levels. Intriguingly, these studies also demonstrated that nuclear beta-catenin staining may not be a sole deciding factor when predicting the status of wnt/beta-catenin signaling in CaP cells. Finally, the extent to which wnt signaling may synergize with an environmental agonist of AhR (2,3,7,8-tetrachlorodibenzo-p-dioxin) to potentiate AhR transcriptional activity was examined. Considering previous work linking AhR to processes of development and carcinogenesis, our data may highlight one particular role for wnt/beta-catenin signaling in prostate tumor biology.

Animals↗
PubMed2003

Two modes of recruitment of E(spl) repressors onto target genes.

The decision of ectodermal cells to adopt the sensory organ precursor fate in Drosophila is controlled by two classes of basic-helix-loop-helix transcription factors: the proneural Ac and Sc activators promote neural fate, whereas the E(spl) repressors suppress it. We show here that E(spl) proteins m7 and mgamma are potent inhibitors of neural fate, even in the presence of excess Sc activity and even when their DNA-binding basic domain has been inactivated. Furthermore, these E(spl) proteins can efficiently repress target genes that lack cognate DNA binding sites, as long as these genes are bound by Ac/Sc activators. This activity of E(spl)m7 and mgamma correlates with their ability to interact with proneural activators, through which they are probably tethered on target enhancers. Analysis of reporter genes and sensory organ (bristle) patterns reveals that, in addition to this indirect recruitment of E(spl) onto enhancers via protein-protein interaction with bound Ac/Sc factors, direct DNA binding of target genes by E(spl) also takes place. Irrespective of whether E(spl) are recruited via direct DNA binding or interaction with proneural proteins, the co-repressor Groucho is always needed for target gene repression.

Animals↗
PubMed2004

Identification of Flk-1 target genes in vasculogenesis: Pim-1 is required for endothelial and mural cell differentiation in vitro.

The tyrosine kinase receptor fetal liver kinase 1 (Flk-1) plays a crucial role in vasculogenesis and angiogenesis, but its target genes remain elusive. Comparing Flk-1(+/+) with Flk-1(-/-) embryonic stem (ES) cells, we identified transcripts regulated by the vascular endothelial growth factor A (VEGF-A)/Flk-1 pathway at an early stage of their differentiation to endothelial and mural precursors. Further analysis of a number of these genes (Nm23-M1, Nm23-M2, Slug, Set, pp32, Cbp, Ship-1, Btk, and Pim-1) showed that their products were transiently up-regulated in vivo in endothelial cells (ECs) during angiogenesis of the ovary, and their mRNA was rapidly induced in vitro by VEGF-A in human umbilical cord vein endothelial cells (HUVECs). Functional analysis by RNA interference (RNAi) in ES cells induced to differentiate demonstrated that Pim-1 is required for their differentiation into ECs and smooth muscle cells (SMCs). In HUVECs, RNAi showed that Pim-1 is required in ECs for VEGF-A-dependent proliferation and migration. The identification of Flk-1 target genes should help in elucidating the molecular pathways that govern the vasculogenesis and angiogenesis processes.

Animals↗
PubMed2005

Phox2 and dHAND transcription factors select shared and unique target genes in the noradrenergic cell type.

The noradrenergic cell type is characterized by the expression of proteins involved in the biosynthesis, transport, and secretion of noradrenaline and is dependent on the sequential and combinatorial expression of numerous transcription factors, including Phox2a, Phox2b, dHAND, GATA2, GATA3, and MASH1. Phox2a and Phox2b transactivate the promoter of the gene encoding the noradrenergic biosynthetic enzyme, dopamine beta-hydroxylase (DBH), and dHAND potentiates the activity of Phox2a. In this study, we use chromatin immunoprecipitation assays to identify target genes of the Phox2 proteins and dHAND. All three proteins are bound to the DBH and PHOX2B promoter regions in SH-SY5Y neuroblastoma cells. The interaction between Phox2a and dHAND is analyzed by fluorescent anisotropy, which demonstrates that dHAND causes an eightfold increase in the affinity of Phox2a for its recognition sites on the DBH promoter region. The Phox2 proteins are not found on the genes encoding other noradrenergic enzymatic or transport proteins but are reciprocally bound to each other's promoters in SH-SY5Y cells. Together with Phox2a and Phox2b, dHAND is bound to the PHOX2B promoter and is also associated with the GATA2 and eHAND genes in the absence of the Phox2 proteins. These results demonstrate the direct interactions of the Phox2 and dHAND transcription factors within a noradrenergic cell type. The Phox2 proteins were found to share all target genes, whereas dHAND binds to genes independently of Phox2a.

Animals↗
PubMed1997

Krüppel target gene knockout participates in the proper innervation of a specific set of Drosophila larval muscles.

Krüppel (Kr) encodes a zinc finger-type transcription factor expressed in spatially and temporally restricted patterns during Drosophila embryogenesis. Molecular targets and the mechanism of Kr action have been studied extensively during the early segmentation process, but only little is known about Kr requirement during later development. We report the identification of a target gene of Kr, termed knockout (ko), isolated by virtue of Krüppel in vitro binding sites. Loss and gain of function experiments show that Kr activity maintains ko expression in a subset of muscles. ko encodes a novel protein expressed in several embryonic tissues including Kr-expressing muscles. Movements of embryos deficient for ko activity are uncoordinated. Their muscle pattern is normal, but the patterns of neuromuscular innervation are specifically disarranged. The results suggest that the Kr target gene ko is required for proper innervation of specific muscles by RP motoneurons.

Amino Acid Sequence↗
PubMed1991

Detection of gene targeting by co-conversion of a single nucleotide change during replacement recombination at the immunoglobulin mu heavy chain locus.

A method is described for detecting targeted events at the mu heavy chain gene which relies on co-conversion (or co-exchange) of a point mutation with a selectable marker contained on a replacement vector. The vector, designed for application to IgM producing hybridomas, contains a single nucleotide change within the region of homology with the target gene which encodes a different allotypic determinant of IgM. In a model system where homologous recombination corrected a defective mu gene, the length of homology between this nucleotide change and the position of the double strand break in the vector was found to have a critical influence on the co-conversion frequency. In the vector design ultimately used for targeting in hybridomas, one in 1000-2000 stable transformants produced IgM with the allotype encoded by the exogenous DNA, and Southern blot analysis confirmed that these were derived by targeted integration. The sensitivity of the screening procedure using a monoclonal antibody specific to this allotype enabled a targeted clone to be detected in a pool of stable transformants when present at a frequency at least as low as one per cent. Several different modifications of the target locus were obtained as a consequence of alternative crossover positions and, in some cases, vector DNA concatenation.

Alanine Transaminase↗
PubMed2004

Tocotrienols activate the steroid and xenobiotic receptor, SXR, and selectively regulate expression of its target genes.

Vitamin E is an essential nutrient with antioxidant activity. Vitamin E is comprised of eight members, alpha-, beta-, gamma-, and delta-tocopherols and alpha-, beta-, gamma-, and delta-tocotrienols. All forms of vitamin E are initially metabolized by omega-oxidation, which is catalyzed by cytochrome P450 enzymes. The steroid and xenobiotic receptor (SXR) is a nuclear receptor that regulates drug clearance in the liver and intestine via induction of genes involved in drug and xenobiotic metabolism. We show here that all four tocotrienols specifically bind to and activate SXR, whereas tocopherols neither bind nor activate. Surprisingly, tocotrienols show tissue-specific induction of SXR target genes, particularly CYP3A4. Tocotrienols up-regulate expression of CYP3A4 but not UDP-glucuronosyltransferase 1A1 (UGT1A1) or multidrug resistance protein-1 (MDR1) in primary hepatocytes. In contrast, tocotrienols induce MDR1 and UGT1A1 but not CYP3A4 expression in intestinal LS180 cells. We found that nuclear receptor corepressor (NCoR) is expressed at relatively high levels in intestinal LS180 cells compared with primary hepatocytes. The unliganded SXR interacts with NCoR, and this interaction is only partially disrupted by tocotrienols. Expression of a dominant-negative NCoR enhanced the ability of tocotrienols to induce CYP3A4 in LS180 cells, suggesting that NCoR plays an important role in tissue-specific gene regulation by SXR. Our findings provide a molecular mechanism explaining how vitamin supplements affect the absorption and effectiveness of drugs. Knowledge of drug-nutrient interactions may help reduce the incidence of decreased drug efficacy.

Cell Line↗
PubMed2006

Epididymis-specific promoter-driven gene targeting: a transcription factor which regulates epididymis-specific gene expression.

Mammalian spermatozoa undergo several modification and finally acquire the ability to fertilize during epididymal transit. One of the distinct features of the epididymis is that it displays a highly regionalized pattern of gene expression. This tissue-, region-, and cell-specific pattern of gene expression is critical for the maintenance of a fully functional epididymis. One would hypothesize that disrupting this process provides an ideal approach to male contraception, since it would not interfere with testicular endocrine output or sperm production. To achieve this purpose, we studied a cluster of epididymis-specific lipocalin genes for understanding the specific mechanisms involved in the control of gene expression in the epididymis. We have identified six epididymis-specific lipocalin genes that are differently regulated and regionalized in the epididymis. Lipocalin 5 [Lcn5 or epididymal retinoic acid-binding protein (E-RABP)] is a member of this epididymis-specific lipocalin gene cluster, which binds hydrophobic molecules such as retinoic acid. We have previously shown that the 5kb promoter fragment of the Lcn5 gene confers both androgen-dependent regulation and epididymis-specific gene expression in transgenic mice whereas 0.6 kb promoter fragment does not. To further narrow down the important cis-regulatory elements that regulate gene expression in the epididymis, we studied the Lcn5 promoter in both transgenic mice and immortalized epididymal cells. We have found that 1.8kb promoter fragment of the Lcn5 gene was sufficient for tissue- and region-specific expression in transgenic mice, and that a transcription factor Forkhead box A2 (Foxa2) interacts with the androgen receptor and binds to the 100 bp fragment of the Lcn5 promoter between 1.2 and 1.3 kb. Our finding provides a framework for further analysis of the epididymal lipocalin gene regulation and modulated control of epididymis-specific expression.

Animals↗
PubMed2006

Frequent epigenetic inactivation of p53 target genes in seminomatous and nonseminomatous germ cell tumors.

Hypermethylation of tumor-suppressor genes has been implicated in the pathogenesis of human cancers. This study was designed to examine the methylation profiles of a selected group of p53 target genes (APAF-1, CASP-8, DAPK-1, IGFBP-3) and to correlate the findings with the histopathological characterization of testicular germ cell tumors (TGCT). Promoter methylation status was analysed by highly sensitive real-time methylation-specific PCR in 46 primary TGCTs (26 seminomas and 20 nonseminomas) and 15 normal testicular tissue samples. APAF-1 methylation was detected in all of the seminomatous and nonseminomatous TGCTs as well as in 60% of normal testicular tissue. Methylation of DAPK-1 was frequent in seminomas (50%) and nonseminomas (20%), but not in normal testicular tissue (6%). The degree of DAPK-1 methylation correlated with the clinical stage of the disease (P=0.05) and was useful in differentiating seminomatous from nonseminomatous, and malignant from nonmalignant testicular tissue (P=0.04 and 0.02, respectively). The APAF-1 methylation index achieved a highly significant differentiation between seminomatous or nonseminomatous tissue and nonmalignant testicular tissue (P=0.0001). In testicular tumorigenesis, promoter methylation of specific p53 target genes occurs at early stage but to varying degrees. Methylation also occurs in normal testicular tissue, which is in contrast to findings in other urogenital malignancies. Further studies will be necessary to determine whether the methylation level may be used as marker for risk estimation, especially in clinical stage I disease.

Apoptosis Regulatory Proteins↗
PubMed2004

Brn-3a transcription factor blocks p53-mediated activation of proapoptotic target genes Noxa and Bax in vitro and in vivo to determine cell fate.

The Brn-3a POU transcription factor is associated with survival and the differentiation of sensory neuronal cells during development. Brn-3a mediates its effects either by the direct regulation of target genes or indirectly upon interaction with proteins such as p53. Brn-3a differentially regulates p53-mediated gene expression and modifies its effect on cell fate. Here we show that, like Bax, Brn-3a antagonizes p53-mediated transcription of another proapoptotic target, Noxa, significantly reducing transactivation of the Noxa promoter by p53. This effect requires the p53 binding site, and electrophoretic mobility shift assay studies suggest that Brn-3a is associated with p53 when it is bound to its site in the Noxa promoter. The wild type but not the mutant promoter can be immunoprecipitated with Brn-3a in chromatin immunoprecipitation assays. Thus, Brn-3a may act by preventing the recruitment of cofactors required for p53 to transactivate this promoter. The co-expression of Brn-3a and p53 results in decreased endogenous Noxa protein in the neuronal cell line, ND7, suggesting a direct functional effect of this interaction. Moreover, there is a significant elevation of both proapoptotic Bax and Noxa proteins in sensory neuronal tissue taken from Brn-3a-/- embryos during development, compared with wild type controls. Striking changes occurred at embryonic day 14.5, a time that precedes a significant loss of specific neurons in the mutant embryos, but not at embryonic day 16.5 when Brn-3a-expressing cells are already lost by apoptosis. Therefore, the lack of antagonism by Brn-3a on activation of proapoptotic p53 target genes may contribute to the increased apoptosis seen in the Brn-3a-/- embryos. These results support a crucial role for Brn-3a in determining the pathway taken by p53 when co-expressed during development and thus in controlling the fate of these cells.

Animals↗
PubMed1999

Highly sensitive detection of target gene by electrochemical method.

A highly sensitive DNA sensing method was developed using electrochemically active ligand. This method is based on the detection of electric current generated by electrochemically active ligand concentrated on the electrode. Electrochemically active, intercalating ligand can bind to the double stranded DNA of target gene sequence on the electrode, where the complementary single strand is immobilized as a probe. We succeeded in the detection of 0.1 amol target gene. The technique was applicable to the detection of 0.1-10 amol gene.

DNA↗
PubMedDate not supplied

Antibody-mediated endocytosis of G250 tumor-associated antigen allows targeted gene transfer to human renal cell carcinoma in vitro.

Specific gene transfer into targeted tumor cells remains a critical issue for the development of systemic gene therapy protocols. With this end in view, we have tested the possibility of selectively directing genes to tumor cells through the recognition of tumor-associated antigens (TAA). This was approached in vitro on four human renal cell carcinoma (RCC) lines by means of the highly specific mouse G250 monoclonal antibody (mAb) chemically conjugated to a plasmid DNA conveying a reporter activity. This mAb directed to a TAA that is present on 95% of primary RCCs and on 60% of metastatic human RCCs was extensively characterized, including during clinical trials. Epifluorescence microscopy analysis indicated that upon specific binding to G250 TAA, G250 mAb alone or conjugated to plasmid DNA was internalized by an active endocytic process and colocalized with the transferrin concentrated in the late recycling perinuclear compartment. We also observed that both unconjugated G250 mAb or G250 mAb conjugated to plasmid DNA remained in the perinuclear region of the cells for > or = 20 hours and were not rapidly translocated to lysosomes or recycled to the plasma membrane. In contrast, unconjugated plasmid DNA was not internalized. After transfection of G250 TAA-positive RCC lines with G250 mAb conjugated to a plasmid cDNA encoding mouse interleukin-2, a significant and sustained production of mouse interleukin-2 protein was detected from days 5-15 and was abrogated by inhibiting the internalization process. Altogether, our data showed that endocytosis of G250 TAA should be the basis of gene transfer to RCC, suggesting that targeting of TAA capable of internalization may be the basis of new approaches for designing alternative cancer gene therapy procedures.

Animals↗