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At least 937 records · Page 52Linked to original sources
PubMed2000

Characterization of the fasting-induced adipose factor FIAF, a novel peroxisome proliferator-activated receptor target gene.

Fasting is associated with significant changes in nutrient metabolism, many of which are governed by transcription factors that regulate the expression of rate-limiting enzymes. One factor that plays an important role in the metabolic response to fasting is the peroxisome proliferator-activated receptor alpha (PPARalpha). To gain more insight into the role of PPARalpha during fasting, and into the regulation of metabolism during fasting in general, a search for unknown PPARalpha target genes was performed. Using subtractive hybridization (SABRE) comparing liver mRNA from wild-type and PPARalpha null mice, we isolated a novel PPARalpha target gene, encoding the secreted protein FIAF (for fasting induced adipose factor), that belongs to the family of fibrinogen/angiopoietin-like proteins. FIAF is predominantly expressed in adipose tissue and is strongly up-regulated by fasting in white adipose tissue and liver. Moreover, FIAF mRNA is decreased in white adipose tissue of PPARgamma +/- mice. FIAF protein can be detected in various tissues and in blood plasma, suggesting that FIAF has an endocrine function. Its plasma abundance is increased by fasting and decreased by chronic high fat feeding. The data suggest that FIAF represents a novel endocrine signal involved in the regulation of metabolism, especially under fasting conditions.

Adipose Tissue↗
PubMed2004

Gene targeting demonstrates that the Plasmodium berghei subtilisin PbSUB2 is essential for red cell invasion and reveals spontaneous genetic recombination events.

The Plasmodium merozoite proteases involved in the crucial process of erythrocyte invasion are promising targets for novel malaria control strategies. We report here the characterization of the subtilisin-like protease SUB2 from the rodent parasites Plasmodium berghei and Plasmodium yoelii, leading the way to in vivo functional studies of this enzyme. The kinetics of expression and subcellular localization imply a central role for SUB2 in erythrocyte invasion. Through the use of gene targeting strategies, we assessed the relevance of P. berghei SUB2 for the intraerythrocytic cycle. The selection of recombinant Pbsub2-TrimycDuoXpress-tagged parasites and the proper expression of the modified coding region demonstrate that the Pbsub2 locus is accessible to genetic modifications. However, Pbsub2 knock-out parasites were not recovered, confirming the importance of PbSUB2 for P. berghei merozoite stages, and supporting the fact that its Plasmodium falciparum SUB2 orthologue is an attractive drug target candidate. Finally, we identify revertant parasites that have lost the integrated selection cassette while conserving a Pbsub2-tagged gene. These spontaneous reversion events should overcome the scarcity of selectable markers available for this parasite, giving access to multiple gene tagging strategies, which, together with the validation of a TrimycDuoXpress tag, would represent valuable new tools for studying the biology of P. berghei.

Amino Acid Sequence↗
PubMed1993

Dominant negative activity of an endogenous thyroid hormone receptor variant (alpha 2) is due to competition for binding sites on target genes.

Regulation of development and metabolism by thyroid hormone (T3) may be influenced by a non-T3 binding T3 receptor (TR) isoform, TR alpha 2, which can inhibit transcriptional activation by legitimate TRs. Numerous mechanisms have been postulated to explain the dominant negative actions of TR alpha 2, including competition for target genes, formation of inactive heterodimers, and squelching. We have found that excess TR alpha 2 was required to inhibit TR alpha 1-mediated transactivation from multiple T3 response elements (TREs). Inhibition of T3 action by TR alpha 2 was specific for TRE-containing genes, because a GAL4/TR alpha 1 chimera, which heterodimerized with the 9-cis-retinoic acid receptor (RXR) and activated transcription from the GAL4 binding site in the presence of T3, was not inhibited by TR alpha 2. In contrast, TR alpha 2 inhibited transactivation by TR alpha/VP16, a chimeric protein containing the N-terminal DNA binding domain (DBD) of TR alpha 1 fused to the transcriptional activation domain of VP16. Indeed, TR alpha 2 inhibited the binding of TR alpha 1 monomers, homodimers, and RXR-heterodimers to DNA in vitro, whereas the TR alpha 2 C terminus alone did not. Although TR alpha 2 bound to TREs with less affinity than TR alpha 1, it bound directly to target genes in the cell nucleus. Furthermore, a TR alpha 2 mutant which binds more avidly to TREs was a more effective inhibitor of T3 action than wild type TR alpha 2. Together these data indicate that TR alpha 2 inhibits T3 action by competing for binding to TREs.

Base Sequence↗
PubMed2006

Identifying target genes of the aryl hydrocarbon receptor nuclear translocator (Arnt) using DNA microarray analysis.

The aryl hydrocarbon receptor nuclear translocator (Arnt) is a basic helix-loop-helix (bHLH) protein that also contains a Per-Arnt-Sim (PAS) domain. In addition to forming heterodimers with many other bHLH-PAS proteins, including the aryl hydrocarbon receptor (AhR) and hypoxia-inducible factors 1alpha, 2alpha and 3alpha, Arnt can also form homodimers when expressed from its cDNA in vitro or in vivo. However, target genes of the Arnt/Arnt homodimer remain to be identified. In this study, we have elucidated the profile of genes responsive to the reintroduction of Arnt expression in an Arnt-deficient mouse hepatoma cell line (c4), using DNA microarray analysis. The expression of 27 genes was upregulated by 1.5-fold or more in c4 cells infected with a retroviral vector expressing mouse Arnt, while no genes were found to be downregulated. Among the upregulated genes, BCL2/adenovirus E1B 19 kDa-interacting protein 1 (NIP3), serine (or cysteine) proteinase inhibitor, clade E, member 1 (PAI1), and N-myc downstream regulated-like (NDR1), were confirmed to be induced by Arnt using real-time PCR. We also found that the 5' promoter region of 15 out of 20 upregulated genes contain the type 2 E-box 5'-CACGTG-3' Arnt/Arnt binding sequence, consistent with the notion that they represent target genes for Arnt.

Animals↗
PubMed2005

Oligonucleotide-based microarray analysis of retinoic acid target genes in the protochordate, Ciona intestinalis.

Oligonucleotide-based microarray analyses were carried out to identify retinoic acid target genes in embryos of the ascidian Ciona intestinalis. Of 21,938 spots, 50 (corresponding to 43 genes) showed over twofold up-regulation in retinoic acid-treated tail bud embryos. In situ hybridization verified retinoic acid-induced up-regulation of 23 genes. Many of them were expressed in the anterior tail region, where a retinaldehyde dehydrogenase homolog is expressed. Homologs of vertebrate genes involved in neurogenesis and/or neuronal functions (e.g., COUP-TF, Ci-Hox1, and SCO-spondin) were expressed in the central nervous system of Ciona embryos, and activated by retinoic acid. Genes encoding transcription factors (e.g., Ci-lmx1.2, vitamin D receptor, and Hox proteins) and apoptosis-related proteins (e.g., transglutaminase and an apoptosis-inducing factor homolog) were also activated by retinoic acid. Simultaneous treatment of embryos with retinoic acid and puromycin revealed a few direct targets, including genes encoding Ci-Hox1, Ci-Cyp26, and an Rnf126-like ring finger protein.

Animals↗
PubMed2003

c-MYC apoptotic function is mediated by NRF-1 target genes.

A detailed understanding of the signaling pathways by which c-Myc elicits apoptosis has proven elusive. In the current study, we have evaluated whether the activation of the mitochondrial apoptotic signaling pathway is linked to c-Myc induction of a subset of genes involved in mitochondrial biogenesis. Cytochrome c and other nuclear-encoded mitochondrial genes are regulated by the transcription factor nuclear respiratory factor-1 (NRF-1). The consensus binding sequence (T/C)GCGCA(C/T)GCGC(A/G) of NRF-1 includes a noncanonical CA(C/T)GCG Myc:MAX binding site. In this study, we establish a link between the induction of NRF-1 target genes and sensitization to apoptosis on serum depletion. We demonstrate, by using Northern analysis, transactivation assays, and in vitro and in vivo promoter binding assays that cytochrome c is a direct target of c-Myc. Like c-Myc, NRF-1 overexpression sensitizes cells to apoptosis on serum depletion. We also demonstrate that selective interference with c-Myc induction of NRF-1 target genes by using a dominant-negative NRF-1 prevented c-Myc-induced apoptosis, without affecting c-Myc-dependent proliferation. These results suggest that c-myc expression leads to mitochondrial dysfunction and apoptosis by deregulating genes involved in mitochondrial function.

3T3 Cells↗
PubMed2003

Engrailed cooperates with extradenticle and homothorax to repress target genes in Drosophila.

Engrailed is a key transcriptional regulator in the nervous system and in the maintenance of developmental boundaries in Drosophila, and its vertebrate homologs regulate brain and limb development. Here, we show that the functions of both of the Hox cofactors Extradenticle and Homothorax play essential roles in repression by Engrailed. Mutations that remove either of them abrogate the ability of Engrailed to repress its target genes in embryos, both cofactors interact directly with Engrailed, and both stimulate repression by Engrailed in cultured cells. We suggest a model in which Engrailed, Extradenticle and Homothorax function as a complex to repress Engrailed target genes. These studies expand the functional requirements for extradenticle and homothorax beyond the Hox proteins to a larger family of non-Hox homeodomain proteins.

Animals↗
PubMed2002

Target gene mutation profile differs between gastrointestinal and endometrial tumors with mismatch repair deficiency.

Mutation frequencies at 25 genes containing coding repeats were compared in colorectal, gastric, and endometrial mismatch repair-deficient (MSI-H) tumors. The overall number of mutations was significantly lower in endometrial than in gastrointestinal MSI-H cancers. Using a likelihood statistical method, target genes were divided in each tumor location into two groups likely to represent gene mutations that do or do not provide selective pressures during tumoral progression. Mutation profiles were quite similar in gastric and colorectal MSI-H cancers but were different in endometrial MSI-H tumors. Deletions in Bat-25 and Bat-26 noncoding repeats were also significantly less important in endometrial as compared with gastrointestinal MSI-H tumors. Our results show that the profile of target gene mutations in MSI-H tumors is tissue specific, with both qualitative and quantitative differences between gastrointestinal and endometrial MSI-H cancers.

Base Pair Mismatch↗
PubMed1999

Identification of avian sarcoplasmic reticulum Ca(2+)-ATPase (SERCA3) as a novel 1,25(OH)(2)D(3) target gene in the monocytic lineage.

Osteoclasts are postmitotic, multinucleated giant cells generated by the fusion of hematopoietic mononuclear precursors from the monocyte-macrophage lineage. In culture, adherent macrophages from blood-derived monocytes grow, gather, and fuse together to form multinucleated osteoclast-like cells. These events are controlled by 1,25(OH)(2)D(3). To sort out new 1,25(OH)(2)D(3) target genes involved in osteoclast differentiation, we have performed an RT-PCR differential display using mRNA from macrophages induced for 10 h by 1,25(OH)(2)D(3) compared to nontreated cells. We have identified a new target gene, a chick ATP-dependent Ca(2+) pump, ChkSERCA3. Although the level of the corresponding transcript increases during the differentiation process from macrophages to osteoclast-like cells, its steady-state level is downregulated by hormone treatment. The action of 1,25(OH)(2)D(3) on the Ca(2+)-ATPase gene expression is independent of de novo protein synthesis and is hormone dose dependent. This expression in adult chick was restricted to the hematopoietic cell lineage, spleen, lung, intestine, and brain, whereas no expression was detected in embryos. The function of the protein can be predicted from its high homology with the other members of the SR ATP-dependent Ca(2+) pump family, i.e., storage and control of cytosolic Ca(2+) directly regulated by 1, 25(OH)(2)D(3), further supporting the critical role for intracellular calcium in highly specialized cells such as osteoclasts.

Amino Acid Sequence↗
PubMed2004

Detection of 91 potential conserved plant microRNAs in Arabidopsis thaliana and Oryza sativa identifies important target genes.

MicroRNAs (miRNAs) are an extensive class of tiny RNA molecules that regulate the expression of target genes by means of complementary base pair interactions. Although the first miRNAs were discovered in Caenorhabditis elegans, >300 miRNAs were recently documented in animals and plants, both by cloning methods and computational predictions. We present a genome-wide computational approach to detect miRNA genes in the Arabidopsis thaliana genome. Our method is based on the conservation of short sequences between the genomes of Arabidopsis and rice (Oryza sativa) and on properties of the secondary structure of the miRNA precursor. The method was fine-tuned to take into account plant-specific properties, such as the variable length of the miRNA precursor sequences. In total, 91 potential miRNA genes were identified, of which 58 had at least one nearly perfect match with an Arabidopsis mRNA, constituting the potential targets of those miRNAs. In addition to already known transcription factors involved in plant development, the targets also comprised genes involved in several other cellular processes, such as sulfur assimilation and ubiquitin-dependent protein degradation. These findings considerably broaden the scope of miRNA functions in plants.

Arabidopsis↗
PubMed1999

High-efficiency gene targeting in Schizosaccharomyces pombe using a modular, PCR-based approach with long tracts of flanking homology.

Bähler et al.(1998) recently described a PCR-based system for the deletion, tagging and overexpression of endogenous genes in the fission yeast Schizosaccharomyces pombe. A small set of PCR primers can be used to generate gene-targeting substrates from each of several modules that differ in the selectable marker (ura4(+) or kanMX6), the presence or absence of specific epitope tags (HA, Myc, GST or GFP), the position in which the epitopes will be inserted (C- or N-terminal), and the presence or absence of a regulatable promoter (the nmt1 promoter). This is a straightforward and powerful system: nine different genes were C-terminal tagged at an average efficiency of 73%, using primers producing only 60-81 bp of homology. In contrast, when studying three transcriptionally-silent genes (rec8(+), rec10(+) and rec11(+)) we obtained an average homologous integration efficiency of 4% for 12 targeting constructs when using primers that contained 80 bp of homology. By using a PCR-based increase in the amount of flanking homology to >/=250 bp, we obtained homologous integration efficiencies of up to 100%. Thus, loci of S. pombe that are refractory to gene targeting when using short tracts of homology can be readily modified by increasing the extent of homology flanking the targeting modules. This straightforward and cost-effective approach might therefore be the one of choice for the modification of S. pombe loci in general and of targeting-refractory loci in particular.

Chromosome Mapping↗
PubMed2001

Mechanisms of differential activation of target gene promoters by p53 hinge domain mutants with impaired apoptotic function.

Suppression of tumor cell growth by p53 results from the activation of both apoptosis and cell cycle arrest functions that have been shown to be separable activities of p53. We report here that some mutants in the p53 hinge domain, a short linker between the DNA binding and tetramerization domains, differentially activated the promoters of p53 target genes and possessed an impaired apoptotic function. Our results indicate that the hinge domain may play an important role in differentially regulating p53 cell cycle arrest and apoptotic functions. However, the mechanisms by which p53 hinge domain mutants differentially activate its target genes, e.g. p21(WAF1/CIP1) and Bax, remain unknown. To investigate the possible mechanisms, recombinant p21(WAF1/CIP1) and Bax promoters were constructed, resulting in rearrangement of the existing p53 binding sites within a given promoter or actually swapping p53 binding sites between the two promoters. Our results suggest that multiple mechanisms of differential transactivation occur, depending on the molecular nature of the relevant hinge domain mutant, such as the possibility that dual separate DNA binding sites in the p21(WAF1/CIP1) promoter are responsible for the selective transactivation activity of p53 hinge domain mutant del300-327, which has a large deletion in the hinge domain. Lack of ideal p53 binding sites in the Bax promoter results in less potent activation than that seen with the p21(WAF1/CIP1) promoter when it is transactivated by hinge domain point mutant mutR306P or short deletion mutant del300-308 proteins. How the single mutation or the short deletion affect the conformation of p53 and consequently the transactivation of the Bax promoter will require further investigation of the relevant p53 protein: DNA-binding domain by NMR and x-ray crystallographic techniques.

Apoptosis↗
PubMed2000

Practical and theoretical issues in gene-targeting studies and their application to behaviour.

Some of the major practical and theoretical issues that are associated with gene-targeting studies in mice are discussed. The availability of sufficient space to house the extensive breeding colonies associated with studies in gene-manipulated mice is an important logistical consideration that requires consideration at an early stage. A practical example is discussed which illustrates some of these issues. Problems associated with disease control and methods of maintaining the health status of valuable colonies are also outlined. Differences in the behavioural phenotype of inbred mouse strains pose important issues for study design and selection of host mouse lines. The results from studies exploring variations in the behavioural phenotype of six common inbred strains are briefly outlined. The impact of phenotypic variation on behavioural studies is considered and the implications for experimental design are discussed.

Animals↗
PubMed1995

Gene therapy of metastatic cancer by in vivo retroviral gene targeting.

We have achieved efficient transduction of tumour metastases in vivo by the vascular delivery of retroviral producer cells. Experimental liver metastases in mice were created by intrasplenic injection of tumour cells into the portal venous circulation. Following the establishment of micrometastases, delivery of retroviral producer cells by the same route with a vector containing the Escherichia coli beta-galactosidase (lacZ) gene demonstrated selective in vivo gene transfer to tumour deposits. By this approach, two retroviral producer cell lines encoding cytokines (IL-4 and IL-2) directed tumoricidal inflammatory responses to established metastases. Cytokine gene targeting inhibited metastasis formation and caused significant overall reduction in tumour burden. These results suggest a novel therapeutic approach for the treatment of disseminated cancer.

3T3 Cells↗
PubMed2002

Stabilization of p53 and transactivation of its target genes in response to replication blockade.

Although it is clear that p53 plays a pivotal role in G1/G2 checkpoints to conserve genomic integrity, its role in S phase checkpoint is less well understood. Recently, it has been reported that p53 is transcriptionally impaired even though it is stabilized during replication blockade. However, the mechanisms underlying this phenomenon are not known. In the present study, it has been shown that p53 accumulates and transactivates its target genes such as p21, gadd45 and bax in response to replication blockade in normal and cancer cells. Lack of transcriptional activation under similar conditions in cells lacking p53 shows that p53-target gene activation during replication blockade is indeed p53-dependent. Further, transactivation of p21 in response to replication blockade by hydroxyurea and aphidicolin is similar to that in response to ionizing radiation except that the latter is more immediate compared to the response to replication blockade. These findings suggest that impairment of transcriptionally active p53 in response to replication blockade is not a general phenomenon.

Aphidicolin↗
PubMed1999

Gene targeting of the KI-KII sequence elements in a model pre-B cell line: effects on germline transcription and rearrangement of the kappa locus.

To study the role of individual sequence elements in the coordinate regulation of rearrangement and germline transcription of the kappa locus, we have (developed a gene targeting system with a mouse model pre-B cell line, 38B9. This line can be induced to initiate K germline transcription and V J rearrangement. Importantly, the effects of gene disruption in the cell line can be analyzed independent of selective pressures that may mask effects in the developing immune system of the mouse. We focused our study on targeting mutation of the endogenous KI KII sequence elements to allow a direct comparison with the same gene disruption reported in mouse studies. Mutations were targeted to one allele, and effects on induced transcription and rearrangement were compared to the remaining wild type allele. Our results show that KI KII mutation has little effect on germline transcription, and reduced the frequency of rearrangement two fold compared to the wild type allele. This report demonstrates that the use of model pre-B cell lines for targeted gene disruption is an attractive alternative to targeting the germline of the mouse.

Animals↗
PubMed2004

Messenger RNA expression of target genes in the urinary sediment of patients with chronic kidney diseases.

BACKGROUND: The degree of renal scarring in kidney biopsy is an important prognostic factor in patients with chronic kidney diseases. We hypothesize that gene expression in the urinary sediment reflects the degree of renal damage. METHODS: We studied 29 patients with chronic kidney disease who underwent kidney biopsy (12 immunoglobulin-A nephropathy and 17 glomerulosclerosis) and 10 healthy controls. The mRNA expressions of a panel of target genes in urinary sediment were measured by real-time quantitative polymerase chain reaction. The results were compared with the degree of histological damage and renal function decline. RESULTS: There were significant differences in the urinary expression of transforming growth factor-beta (TGF-beta), monocyte chemotactic protein-1 (MCP-1) and collagen IV between disease groups and controls. Urinary TGF-beta mRNA expression correlated significantly with estimated glomerular filtration rate (r = -0.412, P = 0.029) and the degree of tubulointerstitial scarring (r = 0.418, P = 0.024). Urinary MCP-1 expression correlated with the degree of glomerulosclerosis (r = 0.450, P = 0.014), but not tubulointerstitial scarring. Urinary MCP-1 expression correlated with its corresponding level by enzyme-linked immunosorbent assay (ELISA) (r = 0.650, P<0.001), but TGF-beta expression did not correlate with its ELISA level. Urinary TGF-beta gene expression correlated with its intra-renal expression in glomeruli (r = 0.701, P<0.001) and tubulointerstitium (r = 0.573, P = 0.001) by immunohistochemistry, while urinary MCP-1 gene expression correlated with its staining in glomeruli (r = 0.576, P = 0.001) but not tubulointerstitium. After 12 months, there was a significant inverse correlation between the rate of renal function decline and urinary expression of connective tissue growth factor (r = -0.471, P = 0.010) and collagen I (r = -0.399, P = 0.032), but not TGF-beta or MCP-1. CONCLUSIONS: Amongst the target genes examined, the mRNA expression of TGF-beta in urinary sediment correlated with renal function, the degree of histological damage and intra-renal level in patients with chronic kidney diseases. Measurement of TGF-beta mRNA expression in urine may be a useful non-invasive tool for assessing the severity of renal damage in patients with chronic kidney diseases.

Adult↗
PubMed2002

Targeted gene disruption reveals the role of cysteinyl leukotriene 1 receptor in the enhanced vascular permeability of mice undergoing acute inflammatory responses.

The cysteinyl leukotrienes (cysLTs), leukotriene (LT) C(4), LTD(4), and LTE(4), are proinflammatory lipid mediators generated in the mouse by hematopoietic cells such as macrophages and mast cells. There are two mouse receptors for the cysLTs, CysLT(1) receptor (CysLT(1)R) and CysLT(2)R, which are 38% homologous and are located on mouse chromosomes X and 14, respectively. To clarify the different roles of the CysLT(1)R and CysLT(2)R in inflammatory responses in vivo, we generated CysLT(1)R-deficient mice by targeted gene disruption. These mice developed normally and were fertile. In an intracellular calcium mobilization assay with fura-2 acetoxymethyl ester, peritoneal macrophages from wild-type littermates, which express both CysLT(1)R and CysLT(2)R, responded substantially to 1 x 10(-6) m LTD(4) and slightly to 1 x 10(-6) m LTC(4), whereas the macrophages from CysLT(1)R-deficient mice did not respond to either LTD(4) or LTC(4). Plasma protein extravasation, but not neutrophil infiltration, was significantly reduced in CysLT(1)R-deficient mice subjected to zymosan A-induced peritoneal inflammation. Plasma protein extravasation was also significantly diminished in CysLT(1)R-deficient mice undergoing IgE-mediated passive cutaneous anaphylaxis as compared with the wild-type mice. Thus, the cysLTs generated in vivo by either monocytes/macrophages or mast cells utilize CysLT(1)R for the response of the microvasculature in acute inflammation.

Animals↗