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At least 811 records · Page 45Linked to original sources
PubMed1999

Role of TNF ligand and receptor family in the lymphoid organogenesis defined by gene targeting.

The molecular basis of lymphoid organogenesis has recently been elucidated using gene-targeted mice. Mice deficient in lymphotoxin-alpha (LT alpha) lack lymph nodes and Peyer's patches. The action of LT alpha in lymphoid organogenesis is mediated mostly by the membrane form of LT by a mechanism independent of TNF receptor I (TNFR-I) or II (TNFR-II). Additionally, follicular dendritic cell (FDC) clusters or germinal centers fail to develop in the spleen of LT alpha-deficient mice. Mice deficient in either TNFR-I or LT beta R also fail to develop splenic FDC clusters and germinal centers, indicating that signaling through both TNFR-I and LT beta R is required for the development of these structures. The mechanisms underlying the defective lymphoid organogenesis in LT alpha-deficient mice, together with a natural mutant strain, alymphoplasia (aly) mice, which manifest a quite similar phenotype to LT alpha-deficient mice, were investigated by generating aggregation chimeras. These studies demonstrate that LT alpha and the aly gene product together control lymphoid organogenesis with a close mechanistic relationship in their biochemical pathways through governing distinct cellular compartments; the former acting as a circulating ligand and the latter as a LT beta R-signaling molecule expressed by the stroma of the lymphoid organs.

Animals↗
PubMed2005

Generation of novel landomycins M and O through targeted gene disruption.

Two genes from Streptomyces cyanogenous S136 that encode the reductase LanZ4 and the hydroxylase LanZ5, which are involved in landomycin A biosynthesis, were characterized by targeted gene inactivation. Analyses of the corresponding mutants as well as complementation experiments have allowed us to show that LanZ4 and LanZ5 are responsible for the unique C-11-hydroxylation that occurs during landomycin biosynthesis. Compounds accumulated by the lanZ4/Z5 mutants are the previously described landomycin F and the new landomycins M and O.

Aminoglycosides↗
PubMed2006

Signaling mechanisms, interaction partners, and target genes of STAT6.

Like other members of the signal transducer and activator of transcription (STAT) family of proteins, STAT6 has a dual role as signaling molecule and transcription factor. STAT6 is tightly connected to IL-4 and IL-13 signaling, and plays a key role in TH2 polarization of the immune system, as studies on knockout mice have illustrated impressively. The last 5 years have yielded many new insights into various aspects of STAT6 signaling. While the canonical view of STAT6 activation and biology remains largely unaltered, significant progress has been made in the identification of factors involved in STAT6 activity and STAT6-mediated gene regulation. About 35 different STAT6 target genes have been identified to date, many of which are involved in TH2-associated processes. This review summarizes the existing data on STAT6. Older landmark studies are discussed, as well as surprising recent additions, like hints on inactive STAT6 dimers and the discovery of novel STAT6 isoforms. There is a particular focus on molecular aspects such as modifications of STAT6 and regulation of STAT6-dependent genes, since studies on these aspects have been particularly fruitful during the last few years.

Animals↗
PubMed2001

Targeted gene knockout of inner arm 1 in Tetrahymena thermophila.

Cilia and flagella contain at least eight different types of dynein arms. It is not entirely clear how the different types of arms are organized along the axoneme. In addition, the role each different type of dynein plays in ciliary or flagellar motility is not known. To initiate studies of dynein organization and function in cilia, we have introduced a mutation into one dynein heavy chain gene (DYH6) in Tetrahymena themophila by targeted gene knockout. We have generated mutant cells that lack wild-type copies of the DYH6 gene. We have shown that the DYH6 gene encodes one heavy chain (HC2) of Tetrahymena 18S dynein and that 18S dynein occupies the I1 position in the ciliary axoneme. We have also shown that Tetrahymena I1 is required for normal motility, normal feeding and normal doubling rate.

Amino Acid Sequence↗
PubMed1995

Autoregulated expression of the yeast INO2 and INO4 helix-loop-helix activator genes effects cooperative regulation on their target genes.

In the yeast Saccharomyces cerevisiae, the phospholipid biosynthetic genes are highly regulated at the transcriptional level in response to the phospholipid precursors inositol and choline. In the absence of inositol and choline (derepressing), the products of the INO2 and INO4 genes form a heteromeric complex which binds to a 10-bp element, upstream activation sequence INO (UASINO), in the promoters of the phospholipid biosynthetic genes to activate their transcription. In the presence of inositol and choline (repressing), the product of the OPI1 gene represses transcription dictated by the UASINO element. Curiously, we identified a UASINO-like element in the promoters of both the INO2 and INO4 genes. The presence of the UASINO element in these two promoters suggested that the mechanism for the inositol-choline response would involved regulating expression of the two activator genes. Using a cat reporter gene, we find that INO2-cat expression was regulated 12-fold in response to inositol and choline but that INO4-cat was constitutively expressed. We further observed that INO2-cat was not expressed in either an ino2 or an ino4 mutant strain and was constitutively overexpressed in an opi1 mutant strain. Expression of the INO4-cat gene was affected only by mutation in the INO4 gene itself. Therefore, INO2-cat transcription is regulated by the products of both the INO2 and INO4 genes whereas INO4 must interact with another protein to activate its own transcription. Our data show that derepression of phospholipid biosynthetic gene expression involves two mechanisms: increasing the levels of the INO2 and INO4 gene products and inactivating the OPI1-mediated repression mechanism. We propose a model suggesting that this dual mechanism of regulation accounts for the observed cooperative stimulation of IN01 and CH01 gene expression (phospholipids biosynthetic genes).

Base Sequence↗
PubMed2002

mRTVP-1, a novel p53 target gene with proapoptotic activities.

We identified a novel mouse gene, mRTVP-1, as a p53 target gene using differential display PCR and extensive promoter analysis. The mRTVP-1 protein has 255 amino acids and differs from the human RTVP-1 (hRTVP-1) protein by two short in-frame deletions of two and nine amino acids. RTVP-1 mRNA was induced in multiple cancer cell lines by adenovirus-mediated delivery of p53 and by gamma irradiation or doxorubicin both in the presence and in the absence of endogenous p53. Analysis of RTVP-1 expression in nontransformed and transformed cells further supported p53-independent gene regulation. Using luciferase reporter and electrophoretic mobility shift assays we identified a p53 binding site within intron 1 of the mRTVP-1 gene. Overexpression of mRTVP-1 or hRTVP-1 induced apoptosis in multiple cancer cell lines including prostate cancer cell lines 148-1PA, 178-2BMA, PC-3, TSU-Pr1, and LNCaP, a human lung cancer cell line, H1299, and two isogenic human colon cancer cell lines, HCT116 p53(+/+) and HCT116 p53(-/-), as demonstrated by annexin V positivity, phase-contrast microscopy, and in selected cases 4',6'-diamidino-2-phenylindole staining and DNA fragmentation. Deletion of the signal peptide from the N terminus of RTVP-1 reduced its apoptotic activities, suggesting that a secreted and soluble form of RTVP-1 may mediate, in part, its proapoptotic activities.

Amino Acid Sequence↗
PubMed2004

Gene expression profiling of potential PPARgamma target genes in mouse aorta.

Diminished activity of peroxisome proliferator-activated receptor-gamma (PPARgamma) may play a role in the pathogenesis of hypertension and vascular dysfunction. To better understand what genes are regulated by PPARgamma, an experimental data set was generated by microarray analysis, in duplicate, of pooled aortic mRNA isolated from mice treated for 21 days with a PPARgamma agonist (rosiglitazone) or vehicle. Of the 12,488 probe sets present on the array (Affymetrix MG-U74Av2), 181 were differentially expressed between groups according to a statistical metric generated using Affymetrix software. A significant correlation was observed between the microarray results and real-time RT-PCR analysis of 39 of these genes. Cluster analysis revealed 3 expression patterns, 29 transcripts of moderate abundance that were decreased (-93%) to very low levels, 106 transcripts that were downregulated (-42%), and 46 transcripts that were upregulated (+70%). Functional groups that were decreased included inflammatory response (-93%, n = 6), immune response (-86%, n = 7), and cytokines (-82%, n = 7). There was an overall upregulation in the oxidoreductase activity group (+47%, n = 9). Individually, six transcripts in this group were increased (+72%), and three were decreased (-34%). Fourteen of the genes map to regions in the rat genome that have been linked to increased blood pressure, and of 142 upstream regions analyzed, sequences resembling the DNA binding site for PPARgamma were identified in 101 of the differentially expressed genes.

Animals↗
PubMed2001

Minireview: tissue-specific versus generalized gene targeting of the igf1 and igf1r genes and their roles in insulin-like growth factor physiology.

The insulin-like growth factors (IGF-I and IGF-II) and the IGF-I receptor are critically important for normal growth and development of the organism. Gene-deletion of these elements has demonstrated that IGF-I is important for both prenatal and postnatal development, whereas IGF-II is important during prenatal stages only. The IGF-I receptor gene-deleted mouse dies at birth apparently as a result of poor muscular development. Utilizing the conditional gene-deletion approach, we have demonstrated that mice lacking the liver IGF-I gene have an approximately 80% reduction in circulating total IGF-I levels. Despite this marked reduction, postnatal growth and development was normal, suggesting that liver IGF-I is not essential for this function. Local tissue IGF-I production was unaffected and may compensate for the lack of the liver IGF-I. Further studies are ongoing to establish the role of the endocrine vs. the autocrine/paracrine IGF-I.

Animals↗
PubMed1996

Midkine as a novel target gene for the Wilms' tumor suppressor gene (WT1).

Midkine (MK) is a heparin-binding growth factor which is strongly expressed during the midgestation period of mouse embryogenesis. Wilms' tumor is an embryonal kidney malignancy in infants, and WT1 has been identified as its tumor suppressor gene. The high expression level of MK in all Wilms' tumor specimens so far examined and the presence of two WT1 elements (5'-GCGGGGGCG-3') in the human MK promoter region led us to examine the possible role of the WT1 gene product in the regulation of MK gene expression. A gel shift assay verified the complex formation between the WT1 gene product and WT1 consensus sequence of MK gene. DNase1 footprint analysis also demonstrated that the downstream WT1 element was protected from DNase1 cleavage by the addition of the WT1 protein. The human MK promoter fused with the chloramphenicol acetyltransferase gene (phMK2.3kCAT) was co-transfected with an effector plasmid containing the WT1 gene into several cell lines. Transient transfection assays showed suppression of the MK promoter by WT1 co-transfection in recipient cells; deletion of the WT1 binding site abolished the suppression. The evidence reported in this study indicates that MK gene is a newly identified WT1 target gene.

3T3 Cells↗
PubMed2000

Formation and repair of heteroduplex DNA on both sides of the double-strand break during mammalian gene targeting.

In this study, we examined homologous recombination in mammalian cells using a gene targeting assay in which the introduction of a double-strand-break (DSB) in the vector-borne region of homology to the chromosome resulted in targeted vector integration. The vector-borne DSB was flanked with small palindromic insertions that, when encompassed within heteroduplex DNA (hDNA) formed during targeted vector integration, were capable of avoiding the activity of the mismatch repair (MMR) system. When used in conjunction with an isolation procedure in which the product(s) of each targeted vector integration event were retained for molecular analysis, information about recombination mechanisms was obtained. The examination of marker segregation patterns in independent recombinants revealed the following, (i) hDNA tracts could form simultaneously on each side of the DSB and in both participating homologous regions. Clonal analysis of sectored recombinants revealed that, in the homologous repeats generated by the recombination event, vector-borne palindrome and chromosomal markers were linked in the expected way in each strand of the hDNA intermediate, (ii) hDNA tracts were subject to MMR processing that occurred on opposite sides of the DSB, and (iii) in the majority of recombinants, the vector-borne marker was replaced with the corresponding marker from the chromosome. Bidirectional hDNA formation and MMR processing of both sides of the DSB are consistent with the double-strand-break repair (DSBR) model of recombination.

Base Sequence↗
PubMed2004

CisOrtho: a program pipeline for genome-wide identification of transcription factor target genes using phylogenetic footprinting.

BACKGROUND: All known genomes code for a large number of transcription factors. It is important to develop methods that will reveal how these transcription factors act on a genome wide level, that is, through what target genes they exert their function. RESULTS: We describe here a program pipeline aimed at identifying transcription factor target genes in whole genomes. Starting from a consensus binding site, represented as a weight matrix, potential sites in a pre-filtered genome are identified and then further filtered by assessing conservation of the putative site in the genome of a related species, a process called phylogenetic footprinting. CisOrtho has been successfully used to identify targets for two homeodomain transcription factors in the genomes of the nematodes Caenorhabditis elegans and Caenorhabditis briggsae. CONCLUSIONS: CisOrtho will identify targets of other nematode transcription factors whose DNA binding specificity is known and can be easily adapted to search other genomes for transcription factor targets.

Animals↗
PubMed2006

Identification of primary target genes of phytochrome signaling. Early transcriptional control during shade avoidance responses in Arabidopsis.

The phytochrome (phy) photoreceptors modulate plant development after perception of light. Upon illumination of etiolated seedlings, phys initiate a transcriptional cascade by directly transducing light signals to the promoters of genes encoding regulators of morphogenesis. In light-grown plants, however, little is known about the transcriptional cascade modulated by phys in response to changes in light. The phy entry points in this cascade are completely unknown. We are particularly interested in the shade avoidance syndrome (SAS). Here we describe a subset of six genes whose expression is rapidly modulated by phys during both deetiolation and SAS in Arabidopsis (Arabidopsis thaliana). Using cycloheximide, we provide evidence that four of these phy rapidly regulated (PAR) genes are direct targets of phy signaling during SAS, revealing these genes as upstream components of the transcriptional cascade. Promoter-beta-glucuronidase fusions confirmed that PAR genes are photoregulated at the transcriptional level. Analysis of gene expression in light signal transduction mutants showed that COP1 and DET1 (but not DET2 or HY5) play a role in modulating PAR expression in response to shade in light-grown seedlings. Moreover, genetic analyses showed that one of the genes identified as a direct target of phy signaling was phy-interacting factor 3-like-1 (PIL1). PIL1 has previously been implicated in SAS in response to transient shade, but we show here that it also plays a key role in response to long-term shade. The action of PIL1 was particularly apparent in a phyB background, suggesting an important negative role for PIL1 under dense vegetation canopies.

Arabidopsis↗
PubMed1992

[Transgenic mouse and gene targeting].

Transgenic mouse technology has proved to be a powerful tool for medical research. So far, a large number of transgenic mice have been generated expressing, e.g. oncogenes, viral genes, immunoglobulins, lymphokines and MHC antigens, and have provided much valuable information. Furthermore, gene targeting technology (homologous recombination between specific chromosomal DNA sequences and exogenously introduced DNA sequences) has been improved and applied to pluripotent, mouse embryonic stem (ES) cells, providing the means to create mice of specifically altered genotype. In this review, some of the background and recent advances of transgenic mouse technology and gene targeting in mouse ES cells are described.

Animals↗
PubMed2003

Egr-1 target genes in human endothelial cells identified by microarray analysis.

Early growth response factor 1 (Egr-1) is a key transcriptional factor to mediate gene expression after vascular injury. To better understand the role of Egr-1 in vasculature, we globally profiled Egr-1 target genes in human endothelial cells using adenoviral gene transfer and Affymetrix oligonucleotide-based microarray technology. More than 300 genes regulated by >/=3-fold with Egr-1 overexpression were identified and, partially, confirmed by Northern and Western blotting, including genes for transcriptional regulators, signaling proteins, cell cycle regulatory proteins, growth factors, and cytokines. Among them, thymus-expressed chemokine (TECK) and IP-30 were dramatically induced by Egr-1, but TNFalpha-related apoptosis inducing ligand (TRAIL) was significantly repressed by Egr-1, suggesting that Egr-1 is a key mediator of inflammation and apoptosis in vascular cells. These data provide novel Egr-1 target genes and contribute to the understanding of the role of Egr-1 in vasculature.

Adenoviridae↗
PubMed2006

Efficient expression of a transgene in platelets using simian immunodeficiency virus-based vector harboring glycoprotein Ibalpha promoter: in vivo model for platelet-targeting gene therapy.

Platelets release several mediators that modify vascular integrity and hemostasis. In the present study, we developed a technique for efficient transgene expression in platelets in vivo and examined whether this targeted-gene-product delivery system using a platelet release reaction could be exploited for clinical applications. Analysis of luciferase reporter gene constructs driven by platelet-specific promoters (the GPIIb, GPIbalpha, and GPVI) revealed that the GPIbalpha promoter was the most potent in the megakaryoblastic cell line UT-7/TPO and human CD34+-derived megakaryocytes. Transduction of UT-7/TPO; CD34+-derived megakaryocytes; and c-Kit+, ScaI+, and Lineage- (KSL) murine hematopoietic stem cells with a simian immunodeficiency virus (SIV)-based lentiviral vector carrying eGFP resulted in efficient, dose-dependent expression of eGFP, and the GPIbalpha promoter seemed to bestow megakaryocytic-specific expression. Transplantation of KSL cells transduced with SIV vector containing eGFP into mice showed that there was preferable expression of eGFP in platelets driven by the GPIbalpha promoter [7-11% for the cytomeglovirus (CMV) promoter, 16-27% for the GPIbalpha promoter]. Furthermore, transplantation of ex vivo-transduced KSL cells by SIV vector carrying human factorVIII (hFVIII) driven by the GPIbalpha promoter induced the production of detectable transcripts of the hFVIII gene and the hFVIII antigen in bone marrow and spleen for at least 90 days and partially corrected the hemophilia A phenotype. Platelet-targeting gene therapy using SIV vectors appears to be promising for gene therapy approaches toward not only inherited platelet diseases but also other hemorrhagic disorders such as hemophilia A.

Animals↗
PubMed1999

Insights into apolipoprotein B biology from transgenic and gene-targeted mice.

Over the past five years, several laboratories have used transgenic and gene-targeted mice to study apolipoprotein (apo) B biology. Genetically modified mice have proven useful for investigating the genetic and environmental factors affecting atherogenesis, for defining apoB structure/function relationships, for understanding the regulation of the apoB gene expression in the intestine, for defining the "physiologic rationale" for the existence of the two different forms of apoB (apoB48 and apoB100) in mammalian metabolism and for providing mechanistic insights into the human apoB deficiency syndrome, familial hypobetalipoproteinemia. This review will provide several examples of how genetically modified mice have contributed to our understanding of apoB biology, including our new discovery that human heart myocytes secrete nascent apoB-containing lipoproteins.

Animals↗
PubMed2004

Disruption of WT1 gene expression and exon 5 splicing following cytotoxic drug treatment: antisense down-regulation of exon 5 alters target gene expression and inhibits cell survival.

Deregulated expression of the Wilms' tumor gene (WT1) has been implicated in the maintenance of a malignant phenotype in leukemias and a wide range of solid tumors through interference with normal signaling in differentiation and apoptotic pathways. Expression of high levels of WT1 is associated with poor prognosis in leukemias and breast cancer. Using real-time (Taqman) reverse transcription-PCR and RNase protection assay, we have shown up-regulation of WT1 expression following cytotoxic treatment of cells exhibiting drug resistance, a phenomenon not seen in sensitive cells. WT1 is subject to alternative splicing involving exon 5 and three amino acids (KTS) at the end of exon 9, producing four major isoforms. Exon 5 splicing was disrupted in all cell lines studied following a cytotoxic insult probably due to increased exon 5 skipping. Disruption of exon 5 splicing may be a proapoptotic signal because specific targeting of WT1 exon 5-containing transcripts using a nuclease-resistant antisense oligonucleotide (ASO) killed HL60 leukemia cells, which were resistant to an ASO targeting all four alternatively spliced transcripts simultaneously. K562 cells were sensitive to both target-specific ASOs. Gene expression profiling following treatment with WT1 exon 5-targeted antisense showed up-regulation of the known WT1 target gene, thrombospondin 1, in HL60 cells, which correlated with cell death. In addition, novel potential WT1 target genes were identified in each cell line. These studies highlight a new layer of complexity in the regulation and function of the WT1 gene product and suggest that antisense directed to WT1 exon 5 might have therapeutic potential.

Alternative Splicing↗