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Results for “ectopic gene conversion”

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At least 217 records · Page 12Linked to original sources

PubMed1997

Drosophila cubitus interruptus forms a negative feedback loop with patched and regulates expression of Hedgehog target genes.

The Drosophila segment polarity gene cubitus interruptus (ci) encodes a zinc finger protein that is required for the proper patterning of segments and imaginal discs. Epistasis analysis indicates that ci functions in the Hedgehog (Hh) signal transduction pathway and is required to maintain wingless expression in the embryo. In this paper, the role of the Ci protein in the Hh signaling pathway is examined in more detail. Our results show that ectopic expression of ci in imaginal discs and the embryo activates the expression of Hh target genes. One of these target genes, patched, forms a negative feedback loop with ci that is regulated by Hh signal transduction. Activation is also achieved using the Ci zinc finger domain fused to a heterologous transactivation domain. Conversely, repression of Hh target genes occurs in animals expressing the Ci zinc finger domain fused to a repression domain. To examine Ci function in more detail, regions of the Ci protein that are responsible for its ability to transactivate and its subcellular distribution have been identified.

Animals↗

PubMed2005

Disruption of p53 by the viral oncoprotein HPV16-E6 does not deregulate chromosomal homologous recombination in a transcriptional interference-free assay system.

In addition to its well established role in the maintenance of genome integrity by regulating transcription of genes involved in cell cycle arrest and programmed cell death, the tumour suppressor p53 has also been shown to inhibit spontaneous chromosomal homologous recombination (HR) between adjacent transcription units, raising the possibility that p53 may prevent chromosomal rearrangements by suppressing HR between repetitive DNA elements (ectopic HR). Consistent with its role in the maintenance of genome integrity is that p53 does not suppress HR between homologous chromosomes (allelic HR) or identical sister chromatids, raising the question of how p53 discriminates between ectopic and allelic HR events. Here, we report that disruption of human p53 by the viral oncoprotein HPV16-E6 does not result in increased rates of chromosomal HR between adjacent DNA repeats in a transcriptional interference-free assay system in which a HR reporter gene can escape transcription repression. These results argue against a direct role for p53 in the regulation of HR mechanisms, imply that HR assay systems may be important determinants of the outcome, and suggest that p53 may suppress ectopic HR through its known ability to repress transcription and alter chromatin structure.

Acetyltransferases↗

PubMed1994

Transcriptional induction of Ty recombination in yeast.

Families of repeated sequences are present in the genomes of all eukaryotes. Little is known about the mechanism(s) that prevents recombination between repeated sequences. In the yeast Saccharomyces cerevisiae, recombination between homologous sequences placed at nonhomologous locations in the genome (ectopic recombination) has been shown to occur at high frequencies for artificially created repeats, but at relatively low frequencies for a natural family of repeated sequences, the Ty family. We have previously shown that a high level of Ty cDNA in the cell causes an increase in the rate of nonreciprocal recombination (gene conversion) of a marked Ty element. In the present study, we show that it is also possible to elevate the rate of recombination of a marked Ty by increasing its transcription. This induction is different from, and acts synergistically to, the one seen upon increased levels of donor Ty cDNA. We show that the induction by transcription does not require the products of the RAD50, RAD51, and RAD57 genes. In contrast, cDNA-mediated recombination is dependent on the product of the RAD51 gene but not on products of the genes RAD50 or RAD57.

DNA Repair↗

PubMed1997

Enhancement of overgrowth by gene interactions in lethal(2)giant discs imaginal discs from Drosophila melanogaster.

Recessive lethal mutations of the lethal(2)giant discs (l(2)gd) and lethal(2)fat (l(2)ft) loci of Drosophila melanogaster cause imaginal disc hyperplasia during a prolonged larval stage. Imaginal discs from l(2)ft l(2)gd or Gl(2)gd double homozygotes show more extensive overgrowth than in either single homozygote, and double homozygous l(2)ft l(2)gd mitotic clones in adult flies show much more overgrowth than is seen in clones homozygous for either l(2)gd or l(2)ft alone. dachsous (ds) also acts as an enhancer of l(2)gd, producing dramatically overgrown discs and causing failure to pupariate in double homozygotes. The comb gap (cg) mutation, which also interacts with ds, greatly enhances the tendency of imaginal discs from l(2)gd larvae to duplicate as they overgrow. If l(2)gd homozygotes are made heterozygous for l(2)ft, then several discs duplicate, indicating that l(2)ft acts an a dominant enhancer of l(2)gd. l(2)ft also acts as a dominant enhancer of l(2)gd, and conversely l(2)gd acts as a dominant modifier of l(2)ft. The enhancement of overgrowth caused by various mutant combinations is accompanied by changes in expression of Decapentaplegic and Wingless. These results show that tumor suppressor genes act in combination to control cell proliferation, and that tissue hyperplasia can be associated with ectopic expression of genes involved in pattern formation.

Animals↗

PubMed2002

Ectopic expression of SUPERMAN suppresses development of petals and stamens.

The floral regulatory gene SUPERMAN (SUP) encodes a C2H2 type zinc finger protein that is required for maintaining boundaries between floral organs in Arabidopsis. It has been proposed that the main function of SUP is to balance cell proliferation in the third and fourth whorl of developing flowers, thereby maintaining the boundaries between the two whorls. To gain further insight into the function of SUP, we have ectopically expressed SUP using the promoter of APETALA1 (AP1), a gene that is initially expressed throughout floral meristems and later becomes restricted to the first and second whorls. Flowers of AP1::SUP plants have fewer floral organs, consistent with an effect of SUP on cell proliferation. In addition, the AP1::SUP transgene caused the conversion of petals to sepals and suppressed the development of stamens. The expression of the B function homeotic gene APETALA3 (AP3) and its regulator UNUSUAL FLORAL ORGANS (UFO) were delayed and reduced in AP1::SUP flowers. However, SUP does not act merely through UFO, as constitutive expression of UFO did not rescue the defects in petal and stamen development in AP1::SUP flowers. Together, these results suggest that SUP has both indirect and direct effects on the expression of B function homeotic genes.

Arabidopsis↗

PubMed2006

Negative regulation of primitive hematopoiesis by the FGF signaling pathway.

Hematopoiesis is controlled by multiple signaling molecules during embryonic and postnatal development. The function of the fibroblast growth factor (FGF) pathway in this process is unclear. Here we show that FGF plays a key role in the regulation of primitive hematopoiesis in chicks. Using hemoglobin mRNA expression as a sensitive marker, we demonstrate that timing of blood differentiation can be separated from that of initial mesoderm patterning and subsequent migration. High FGF activity inhibits primitive blood differentiation and promotes endothelial cell fate. Conversely, inhibition of FGFR activity leads to ectopic blood formation and down-regulation of endothelial markers. Expression and functional analyses indicate that FGFR2 is the key receptor mediating these effects. The FGF pathway regulates primitive hematopoiesis by modulating Gata1 expression level and activity. We propose that the FGF pathway mediates repression of globin gene expression and that its removal is essential before terminal differentiation can occur.

Animals↗

PubMed2003

Melanoma differentiation associated gene-7, mda-7/interleukin-24, induces apoptosis in prostate cancer cells by promoting mitochondrial dysfunction and inducing reactive oxygen species.

Mda-7/IL-24 (Ad.mda-7) is a novel cytokine gene belonging to the interleukin (IL) 10 gene superfamily. Adenoviral-mediated delivery of mda-7/IL-24 causes growth suppression and apoptosis in a wide spectrum of cancer cells, including prostate, without harming normal cells. We now demonstrate that Ad.mda-7 selectively induces apoptosis in prostate cancer cells by promoting mitochondrial dysfunction and reactive oxygen species (ROS) production. Antioxidants (N-acetyl-L-cysteine and Tiron) and inhibitors of mitochondrial permeability transition (cyclosporine A and bongkrekic acid) inhibit Ad.mda-7-induced mitochondrial dysfunction and apoptosis. Conversely, agents augmenting ROS production (arsenic trioxide, NSC656240, and PK11195) facilitate Ad.mda-7-induced apoptosis. Ectopic expression of Bcl-2 and Bcl-x(L) inhibits mitochondrial changes, ROS production, and apoptosis providing additional support for an association between mitochondrial dysfunction and Ad.mda-7 action. These studies present definitive evidence that changes in mitochondrial function and ROS production are key components associated with selective killing of prostate cancer cells by mda-7/IL-24.

Adenoviridae↗

PubMed1997

Determination of Arabidopsis floral meristem identity by AGAMOUS.

Determinate growth of floral meristems in Arabidopsis requires the function of the floral regulatory gene AGAMOUS (AG). Expression of AG mRNA in the central region of floral meristems relies on the partially overlapping functions of the LEAFY (LFY) and APETALA1 (AP1) genes, which promote initial floral meristem identity. Here, we provide evidence that AG function is required for the final definition of floral meristem identity and that constitutive AG function can promote, independent of LFY and AP1 functions, the determinate floral state in the center of reproductive meristems. Loss-of-function analysis showed that the indeterminate central region of the ag mutant floral meristem undergoes conversion to an inflorescence meristem when long-day-dependent flowering stimulus is removed. Furthermore, gain-of-function analysis demonstrated that ectopic AG function results in precocious flowering and the formation of terminal flowers at apices of both the primary inflorescence and axillary branches of transgenic Arabidopsis plants in which AG expression is under the control of the 35S promoter from cauliflower mosaic virus. Similar phenotypes were also observed in lfy ap1 double mutants carrying a 35S-AG transgene. Together, these results indicate that AG is a principal developmental switch that controls the transition of meristem activity from indeterminate to determinate.

AGAMOUS Protein, Arabidopsis↗

PubMed1996

Function of ets genes is conserved between vertebrates and Drosophila.

The Drosophila pointed gene encodes two ETS transcriptional activators, pointedP1 and pointedP2, sharing a common C-terminal ETS domain. In the embryonic central nervous system pointedP2 is required for midline glial cell differentiation, whereas, in the eye, pointedP2 is essential for photoreceptor cell differentiation. Both vertebrate c-ets-1 and c-ets-2 gene ETS domains are highly homologous to the one of pointed. In addition, the N-terminal region of pointedP2 and vertebrate ets products share another homologous domain, the so-called RII/pointed box which appears to mediate the ras-dependent phosphorylation/stimulation. Here, we show that the vertebrate ets genes are functionally homologous to the Drosophila pointed gene. pointedP2 efficiently binds to an optimized c-Ets-1/c-Ets-2 probe in vitro, and stimulates two distinct c-Ets-1/c-Ets-2-responsive sequences when transiently expressed in vertebrate cells. Conversely, when vertebrate ets transgenes are expressed during fly development, they are capable of rescuing the pointed mutant phenotype in both midline glia and photoreceptor development. As ectopically expressed pointedP1 can also rescue pointedP2 deficiency in photoreceptor development, it appears that the ability of ets products to phenocopy each other in vivo does not require the conserved RII/pointed box, but rather, primarily relies on the presence of the highly conserved ETS domain.

Animals↗

PubMed2003

p53 mutated in the transactivation domain retains regulatory functions in homology-directed double-strand break repair.

The tumor suppressor p53 transcriptionally transactivates cellular target genes that are implicated in growth control, apoptosis, and DNA repair. However, several studies involving p53 core domain mutants suggested that regulatory functions in recombinative repair do not require transcriptional transactivation and are separable from growth-regulation and apoptosis. Leu22 and Trp23 within the transactivation domain of human p53 play a critical role in binding basal components of the transcription machinery and, therefore, in the transactivation activity of p53. To further delineate whether p53 target genes are involved in recombination regulation, we ectopically expressed p53(22Q,23S) in p53-negative cell lines, which carry reporter systems for different homology-directed double-strand break (DSB) repair events. Like wild-type p53, p53(22Q,23S) efficiently downregulated homologous recombination on two chromosomally integrated substrates without affecting exchange on a substrate for the compound pathway of gene conversion and nonhomologous end joining. Only upon lowering the p53 protein to DNA substrate ratio by several orders of magnitude, we noticed a weak defect of a p53 transactivation domain mutant in DSB repair assays. In conclusion, molecular interactions of p53 within the N-terminal domain are not required to restrain DNA recombination, but might contribute to this genome stabilizing function.

Apoptosis↗

PubMed2006

Induction of PPARbeta and prostacyclin (PGI2) synthesis by Raf signaling: failure of PGI2 to activate PPARbeta.

A role for the nuclear receptor peroxisome proliferator-activated receptor-beta (PPARbeta) in oncogenesis has been suggested by a number of observations but its precise role remains elusive. Prostaglandin I2 (PGI2, prostacyclin), a major arachidonic acid (AA) derived cyclooxygenase (Cox) product, has been proposed as a PPARbeta agonist. Here, we show that the 4-hydroxytamoxifen (4-OHT) mediated activation of a C-Raf-estrogen receptor fusion protein leads to the induction of both the PPARbeta and Cox-2 genes, concomitant with a dramatic increase in PGI2 synthesis. Surprisingly, however, 4-OHT failed to activate PPARbeta transcriptional activity, indicating that PGI2 is insufficient for PPARbeta activation. In agreement with this conclusion, the overexpression of ectopic Cox-2 and PGI2 synthase (PGIS) resulted in massive PGI2 synthesis but did not activate the transcriptional activity of PPARbeta. Conversely, inhibition of PGIS blocked PGI2 synthesis but did not affect the AA mediated activation of PPARbeta. Our data obtained with four different cell types and different experimental strategies do not support the prevailing opinion that PGI2 plays a significant role in the regulation of PPARbeta.

Animals↗

PubMed1998

p300/CREB-binding protein enhances the prolactin-mediated transcriptional induction through direct interaction with the transactivation domain of Stat5, but does not participate in the Stat5-mediated suppression of the glucocorticoid response.

Stat5 was discovered as a PRL-induced member of the Stat (signal transducer and activator of transcription) family. Its induction by many other cytokines and interleukins suggests that Stat5 plays a crucial role not only in mammary epithelial, but also in hematopoietic cells. Cell type- and promoter-specific functions of Stat5 are most likely modulated by the interaction with other transcription factors. We recently showed cross-talk between Stat5 and the glucocorticoid receptor. The activated glucocorticoid receptor forms a complex with Stat5 and enhances Stat5-mediated transcriptional induction. Conversely, Stat5 diminishes the induction of glucocorticoid-responsive genes. Here, we investigated the role of p300/CBP(CREB-binding protein), a transcriptional coactivator of several groups of transcription factors, in Stat5-mediated transactivation and in the cross-talk between Stat5 and the glucocorticoid receptor. p300/ CBP acts as a coactivator of Stat5. Its ectopic expression enhances PRL-induced Stat5-mediated transcriptional activation. Consistent with this observation, we find that the adenovirus E1A protein, which binds to p300/CBP, suppresses Stat5-induced transcriptional activation. This inhibition requires the Stat5 transactivation domain and the p300/CBP binding site of E1A. Coimmunoprecipitation and mammalian two-hybrid assays demonstrate a direct interaction between the carboxyl-terminal transactivation domain of Stat5 and p300/CBP. p300/CBP also positively interacts with the glucocorticoid receptor and enhances glucocorticoid receptor-dependent transcriptional activation of the mouse mammary tumor virus-long terminal repeat promoter. Overexpression of p300/CBP does not counteract the Stat5-mediated inhibition of glucocorticoid receptor-dependent transactivation, i.e. the repression of the glucocorticoid response by Stat5 is not a consequence of competition for limiting amounts of p300/CBP.

Adenovirus E1A Proteins↗

PubMed2002

Down-regulation of TM29, a tomato SEPALLATA homolog, causes parthenocarpic fruit development and floral reversion.

We have characterized the tomato (Lycopersicon esculentum Mill.) MADS box gene TM29 that shared a high amino acid sequence homology to the Arabidopsis SEP1, 2, and 3 (SEPALLATA1, 2, and 3) genes. TM29 showed similar expression profiles to SEP1, with accumulation of mRNA in the primordia of all four whorls of floral organs. In addition, TM29 mRNA was detected in inflorescence and vegetative meristems. To understand TM29 function, we produced transgenic tomato plants in which TM29 expression was down-regulated by either cosuppression or antisense techniques. These transgenic plants produced aberrant flowers with morphogenetic alterations in the organs of the inner three whorls. Petals and stamens were green rather than yellow, suggesting a partial conversion to a sepalloid identity. Stamens and ovaries were infertile, with the later developing into parthenocarpic fruit. Ectopic shoots with partially developed leaves and secondary flowers emerged from the fruit. These shoots resembled the primary transgenic flowers and continued to produce parthenocarpic fruit and additional ectopic shoots. Based on the temporal and spatial expression pattern and transgenic phenotypes, we propose that TM29 functions in floral organ development, fruit development, and maintenance of floral meristem identity in tomato.

Amino Acid Sequence↗

PubMed2006

Connexin 26 regulates epidermal barrier and wound remodeling and promotes psoriasiform response.

Inflammatory skin disorders result in significant epidermal changes, including keratinocyte hyperproliferation, incomplete differentiation, and impaired barrier. Here we test whether, conversely, an impaired epidermal barrier can promote an inflammatory response. Mice lacking the transcription factor Kruppel-like factor 4 (Klf4) have a severe defect in epidermal barrier acquisition. Transcription profiling of Klf4(-/-) newborn skin revealed similar changes in gene expression to involved psoriatic plaques, including a significant upregulation of the gap junction protein connexin 26 (Cx26). Ectopic expression of Cx26 from the epidermis-specific involucrin (INV) promoter (INV-Cx26) demonstrated that downregulation of Cx26 is required for barrier acquisition during development. In juvenile and adult mice, persistent Cx26 expression kept wounded epidermis in a hyperproliferative state, blocked the transition to remodeling, and led to an infiltration of immune cells. Mechanistically, ectopic expression of Cx26 in keratinocytes resulted in increased ATP release, which delayed epidermal barrier recovery and promoted an inflammatory response in resident immune cells. These results provide a molecular link between barrier acquisition in utero and epidermal remodeling after wounding. More generally, these studies suggest that the most effective treatments for inflammatory skin disorders might concomitantly suppress the immune response and enhance epidermal differentiation to restore the barrier.

Animals↗

PubMed1995

Requirement for Drosophila cytoplasmic tropomyosin in oskar mRNA localization.

The localization of oskar (osk) RNA to the posterior pole of the developing fruit fly (Drosophila) oocyte induces the assembly of pole plasm, causing development of the abdomen and germ line. Failure to localize oskar RNA results in embryos that lack abdomen and germ cells. Conversely, mis-targeting of oskar RNA to the anterior of the oocyte causes formation of ectopic abdomen and germ cells at the anterior pole. Maternal mutants that have reduced pole plasm activity produce sterile adults with normal abdominal development, suggesting that germ cells are more sensitive than abdomen to defects in pole plasm assembly. Thus mutations in genes that reduce oskar RNA localization or activity can be recovered as viable sterile adults. In a screen for mutants defective in germ cell formation, we isolated nine alleles of the tropomyosin II gene. Here we show that mutations in tropomyosin II (TmII) virtually abolish oskar RNA localization to the posterior pole, suggesting an involvement of the actin network in oskar RNA localization.

Actins↗

PubMed2005

Genetic characterization of Pseudomonas fluorescens SBW25 rsp gene expression in the phytosphere and in vitro.

The plant-colonizing Pseudomonas fluorescens strain SBW25 harbors a gene cluster (rsp) whose products show similarity to type III protein secretion systems found in plant and animal pathogens. Here we report a detailed analysis of the expression and regulation of the P. fluorescens rsp pathway, both in the phytosphere and in vitro. A combination of chromosomally integrated transcriptional reporter fusions, overexpressed regulatory genes, and specific mutants reveal that promoters controlling expression of rsp are actively transcribed in the plant rhizosphere but not (with the exception of the rspC promoter) in the phyllosphere. In synthetic medium, regulatory (rspL and rspR) and structural (rspU, plus the putative effector ropE) genes are poorly expressed; the rspC promoter is subject to an additional level of regulatory control. Ectopic expression of regulatory genes in wild-type and mutant backgrounds showed that RspR controls transcription of the alternate sigma factor, rspL, and that RspL controls expression of gene clusters encoding structural genes. Mutation of rspV did not affect RspR-mediated expression of rspU. A search for additional regulators revealed two candidates--one with a role in the conversion of alanine to pyruvate--suggesting that expression of rsp is partly dependent upon the metabolic status of the cell. Mutations in rsp regulators resulted in a significant reduction in competitive colonization of the root tips of sugar beet seedlings but also caused a marked increase in the lag phase of laboratory-grown cultures, indicating that rsp regulatory genes play a more significant general role in the function of P. fluorescens SBW25 than previously appreciated.

Arabidopsis↗

PubMed1997

Conversion of ectoderm into a neural fate by ATH-3, a vertebrate basic helix-loop-helix gene homologous to Drosophila proneural gene atonal.

We have isolated a novel basic helix-loop-helix (bHLH) gene homologous to the Drosophila proneural gene atonal, termed ATH-3, from Xenopus and mouse. ATH-3 is expressed in the developing nervous system, with high levels of expression in the brain, retina and cranial ganglions. Injection of ATH-3 RNA into Xenopus embryos dramatically expands the neural tube and induces ectopic neural tissues in the epidermis but inhibits non-neural development. This ATH-3-induced neural hyperplasia does not require cell division, indicating that surrounding cells which are normally non-neural types adopt a neural fate. In a Xenopus animal cap assay, ATH-3 is able to convert ectodermal cells into neurons expressing anterior markers without inducing mesoderm. Interestingly, a single amino acid change from Ser to Asp in the basic region, which mimics phosphorylation of Ser, severely impairs the anterior marker-inducing ability without affecting general neurogenic activities. These results provide evidence that ATH-3 can directly convert non-neural or undetermined cells into a neural fate, and suggest that the Ser residue in the basic region may be critical for the regulation of ATH-3 activity by phosphorylation.

Amino Acid Sequence↗

PubMed2004

Ectopic expression of the petunia MADS box gene UNSHAVEN accelerates flowering and confers leaf-like characteristics to floral organs in a dominant-negative manner.

Several genes belonging to the MADS box transcription factor family have been shown to be involved in the transition from vegetative to reproductive growth. The Petunia hybrida MADS box gene UNSHAVEN (UNS) shares sequence similarity with the Arabidopsis thaliana flowering gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1, is expressed in vegetative tissues, and is downregulated upon floral initiation and the formation of floral meristems. To understand the role of UNS in the flowering process, knockout mutants were identified and UNS was expressed ectopically in petunia and Arabidopsis. No phenotype was observed in petunia plants in which UNS was disrupted by transposon insertion, indicating that its function is redundant. Constitutive expression of UNS leads to an acceleration of flowering and to the unshaven floral phenotype, which is characterized by ectopic trichome formation on floral organs and conversion of petals into organs with leaf-like features. The same floral phenotype, accompanied by a delay in flowering, was obtained when a truncated version of UNS, lacking the MADS box domain, was introduced. We demonstrated that the truncated protein is not translocated to the nucleus. Using the overexpression approach with both the full-length and the nonfunctional truncated UNS protein, we could distinguish between phenotypic alterations because of a dominant-negative action of the protein and because of its native function in promoting floral transition.

Arabidopsis↗