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Results for “Enhancer Elements, Genetic”

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PubMed1994

Genetic and phenotypic analysis of thirteen essential genes in cytological interval 22F1-2; 23B1-2 reveals novel genes required for neural development in Drosophila.

In an attempt to identify mutations in the Drosophila synaptotagmin gene we have isolated many new rearrangements, point mutations and P element insertions in the 22F1-2; 23B1-2 cytological interval on chromosome arm 2L. This interval encompasses 13 cytological bands and is shown to contain 13 essential complementation groups, including decapentaplegic, synaptotagmin and Curly. Through chemical and P element mutagenesis we have isolated seven new deletions, which combined with previously isolated rearrangements, have allowed us to order most genes in the interval. A genomic walk covering approximately 100 kb within this interval spans at least five essential genes as identified by chromosomal aberrations. Preliminary phenotypic characterizations of the mutant phenotype and lethal phase is presented for many mutations. Three loci within this interval are shown to be required for proper neural development. Given that the average number of alleles per complementation group is greater than seven, it is very likely that all essential genes within this cytological interval have been identified.

Alleles↗

PubMed2003

Exonic splicing enhancer-dependent selection of the bovine papillomavirus type 1 nucleotide 3225 3' splice site can be rescued in a cell lacking splicing factor ASF/SF2 through activation of the phosphatidylinositol 3-kinase/Akt pathway.

Bovine papillomavirus type 1 (BPV-1) late pre-mRNAs are spliced in keratinocytes in a differentiation-specific manner: the late leader 5' splice site alternatively splices to a proximal 3' splice site (at nucleotide 3225) to express L2 or to a distal 3' splice site (at nucleotide 3605) to express L1. Two exonic splicing enhancers, each containing two ASF/SF2 (alternative splicing factor/splicing factor 2) binding sites, are located between the two 3' splice sites and have been identified as regulating alternative 3' splice site usage. The present report demonstrates for the first time that ASF/SF2 is required under physiological conditions for the expression of BPV-1 late RNAs and for selection of the proximal 3' splice site for BPV-1 RNA splicing in DT40-ASF cells, a genetically engineered chicken B-cell line that expresses only human ASF/SF2 controlled by a tetracycline-repressible promoter. Depletion of ASF/SF2 from the cells by tetracycline greatly decreased viral RNA expression and RNA splicing at the proximal 3' splice site while increasing use of the distal 3' splice site in the remaining viral RNAs. Activation of cells lacking ASF/SF2 through anti-immunoglobulin M-B-cell receptor cross-linking rescued viral RNA expression and splicing at the proximal 3' splice site and enhanced Akt phosphorylation and expression of the phosphorylated serine/arginine-rich (SR) proteins SRp30s (especially SC35) and SRp40. Treatment with wortmannin, a specific phosphatidylinositol 3-kinase/Akt kinase inhibitor, completely blocked the activation-induced activities. ASF/SF2 thus plays an important role in viral RNA expression and splicing at the proximal 3' splice site, but activation-rescued viral RNA expression and splicing in ASF/SF2-depleted cells is mediated through the phosphatidylinositol 3-kinase/Akt pathway and is associated with the enhanced expression of other SR proteins.

Alternative Splicing↗

PubMed1994

Isolation of the human LIM/homeodomain gene islet-1 and identification of a simple sequence repeat polymorphism [corrected].

The islet-1 (Isl-1) gene encodes a protein that binds to the enhancer region of the insulin gene. Isl-1 is a member of the LIM/homeodomain family of transcription factors. Because insulin deficiency, either relative or absolute, is a cardinal feature of non-insulin-dependent diabetes mellitus (NIDDM), this study addressed the question of whether mutations in genes that regulate insulin production could be involved. Rat Isl-1 was the first insulin enhancer binding protein to be isolated, and, in this study, the rat gene was used to isolate a partial human islet Isl-1 cDNA and subsequently to isolate genomic clones. A simple sequence repeat was found in the Isl-1 gene, and polymerase chain reaction amplification of this region of genomic DNA revealed 12 alleles in St. Louis African-Americans (het = 0.87), 14 alleles in black Nigerians (het = 0.89), 8 alleles in Japanese (het = 0.69), and 8 alleles in Caucasians (het = 0.81). Genetic linkage analysis uniquely placed Isl-1 on chromosome 5q (D5S395[12.8 cM]Isl-1 [11.6 cM]D5S407). The simple sequence repeat polymorphism at the Isl-1 locus was used to evaluate mutations in this gene as a possible contributor to the pathogenesis of NIDDM. Allelic frequencies did not differ between patients with NIDDM (n = 165) and nondiabetic control subjects (n = 163) in two black populations (St. Louis African-Americans and Nigerians). Linkage analyses in 15 nonglucokinase maturity-onset diabetes of the young pedigrees indicated that linkage could be rejected (LOD score < -3.0) over a distance of 15 cM.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗

PubMed2004

Mef2c is activated directly by Ets transcription factors through an evolutionarily conserved endothelial cell-specific enhancer.

Members of the Myocyte Enhancer Factor 2 (MEF2) family of transcription factors play key roles in the development and differentiation of numerous cell types during mammalian development, including the vascular endothelium. Mef2c is expressed very early in the development of the endothelium, and genetic studies in mice have demonstrated that mef2c is required for vascular development. However, the transcriptional pathways involving MEF2C during endothelial cell development have not been defined. As a first step towards identifying the transcriptional factors upstream of MEF2C in the vascular endothelium, we screened for transcriptional enhancers from the mouse mef2c gene that regulate vascular expression in vivo. In this study, we identified a transcriptional enhancer from the mouse mef2c gene sufficient to direct expression to the vascular endothelium in transgenic embryos. This enhancer is active in endothelial cells within the developing vascular system from very early stages in vasculogenesis, and the enhancer remains robustly active in the vascular endothelium during embryogenesis and in adulthood. This mef2c endothelial cell enhancer contains four perfect consensus Ets transcription factor binding sites that are efficiently bound by Ets-1 protein in vitro and are required for enhancer function in transgenic embryos. Thus, these studies identify mef2c as a direct transcriptional target of Ets factors via an evolutionarily conserved transcriptional enhancer and establish a direct link between these two early regulators of vascular gene expression during endothelial cell development in vivo.

Animals↗

PubMed1996

Direct and indirect mechanisms of repression participate in suppression of T-cell-specific gene expression in T x L-cell hybrids.

Expression of tissue-specific genes can be altered upon fusion of mammalian cells of different types. To resolve the genetic basis of this phenomenon and to identify components of the regulatory circuits that are involved, we have established a series of somatic cell hybrids between mouse T cells and L cells. These hybrids have an unusual and interesting phenotype. Unlike many hybrid cells studied, in which the expression of an entire set of tissue-specific genes was coordinately extinguished, in our T x L-cell hybrids only two out of seven T-cell-restricted genes were completely extinguished, whereas the other genes were repressed to various degrees. These hybrids extinguish the production of TCR beta and Thy-1 mRNA, repress the expression of TCR alpha, GATA-3, TCF-1, and LEF-1 genes to different extents, exhibit small changes in the level of CD3-epsilon mRNA, and continue to express the fibroblast-specific fibronectin gene, and the ets-1 gene. In this study we have evaluated for the first time the molecular mechanisms that underlie the repression of TCR alpha and TCR beta chain genes in T x L-cell hybrids. We have shown that multiple repression mechanisms, both direct and indirect, contribute to TCR alpha and TCR beta suppression. Repression of the expression of these genes correlated not only with the downregulation of GATA-3, TCF-1, and LEF-1 transcription factor expression, and with a change in the chromatin structure, but more importantly, with the activation of the silencer activity. Our study provides evidence for the existence of at least two negatively regulating elements, located at the TCR alpha enhancer-containing fragment and at the silencer region, which are active in our hybrid cells. We have shown that there was no correlation between the levels of GATA-3, TCF-1, and LEF-1 expression versus the level of TCR alpha mRNA in the independent hybrids. In contrast, both the silencer activity and the ability of the TCR alpha enhancer to downregulate thymidine kinase (TK) promoter activity were found to be in an inverse correlation with the ability of the different hybrid cells to express TCR alpha mRNA.

Animals↗

PubMed1998

Cdc34 and the F-box protein Met30 are required for degradation of the Cdk-inhibitory kinase Swe1.

Ubiquitin-mediated proteolysis controls the abundance of many cell cycle regulatory proteins. Recent work in Saccharomyces cerevisiae suggests that a complex consisting of Cdc53, Skp1, and a third component known as an F-box protein (termed SCF) in combination with Cdc34 specifically targets regulatory proteins for degradation, and that substrate specificity is likely to be mediated by the F-box subunit. A screen for genetic interactions with a cdc34 mutation yielded MET30, which encodes an F-box protein. MET30 is an essential gene required for cell cycle progression and met30 mutations interact genetically with mutations in SCF components. Furthermore, physical interactions between Met30, Cdc53, Cdc34, and Skp1 in vivo provide evidence for an SCFMet30 complex. We demonstrate the involvement of Met30 in the degradation of the Cdk-inhibitory kinase Swe1. Swe1 is stabilized in met30 mutants and GST-Met30 pull-down experiments reveal that Met30 specifically binds Swe1 in vivo. Furthermore, extracts prepared from cdc34 or met30 mutants are defective in polyubiquitination of Swe1. Taken together, these data suggest that SCF-mediated proteolysis may contribute to the regulation of entry into mitosis. Our data, in combination with previously published results, also provide evidence for distinct SCF complexes in vivo and support the idea that their F-box subunits mediate SCF substrate specificity.

Amino Acid Sequence↗

PubMedDate not supplied

Dissociation of transforming and trans-activation functions for bovine papillomavirus type 1.

It has been shown that genetic information encoded by the 3' open reading frames (ORFs), E2, E3, E4 and E5, of bovine papillomavirus type 1 (BPV-1), is sufficient to induce cellular transformation of certain mouse cells. The product of the E2 ORF has further been shown to be responsible for the trans-activation of a transcriptional regulatory element located in the noncoding region (NCR) of the BPV-1 genome. To examine whether or not the E2 trans-activation function is encoded by the same gene that encodes the 3' ORF viral transformation function, we have now analysed the expression of the trans-activation function in series of mouse C127 cells transformed by BPV-1 deletion mutants. In addition, using mutated complementary DNA clones generated by the insertion of a premature translational termination linker into different sites of a BPV-1 cDNA clone containing the 3' ORFs intact, we demonstrate that transformation and transcriptional trans-activation functions can be dissociated and that they map respectively to the E5 and E2 ORFs.

Acetyltransferases↗

PubMed1992

In vitro transcription faithfully reflecting T-cell activation requirements.

T-cell activation is a complex process mediated by cell membrane molecules including the T-cell antigen receptor (TCR), adhesive molecules, and cytokine receptors that collectively produce an increase in intracellular Ca2+, and activation of protein kinase C that initiate a genetic program resulting in immunologic function and irreversible differentiation. To understand how these cell membrane events are translated into a genetic regulatory cascade resulting in T-cell function, we have developed an in vitro transcription system, derived from Jurkat T-cells, which demonstrates inducible, cell-type-specific transcription following T-cell stimulation. Nuclear extracts from cells stimulated with phorbol 12-myristate 13-acetate and ionomycin, which activate protein kinase C and mimic physiological activation through the T-cell antigen receptor, transcribe an interleukin-2 (IL-2) enhancer (-326 to +24) template 5-fold more efficient than nuclear extracts from resting T-cells and severalfold more efficient than extracts from Jurkat cells treated with phorbol 12-myristate 13-acetate or ionomycin alone. Further results demonstrate that in vitro transcription of the IL-2 enhancer is T-cell specific since nuclear extracts from rat liver and stimulated HeLa cells are unable to induce IL-2 transcription. The activation-dependent, T-cell-specific in vitro transcription system described here should facilitate the dissection of signals that emanate from the T-cell surface resulting in IL-2 transcription and T-cell activation.

Cells, Cultured↗

PubMed1995

Transposon tagging of the maize Glossy2 locus with the transposable element En/Spm.

The Glossy2 (Gl2) locus of maize is required for the formation of the epicuticular wax layer of young plants. gl2 mutant seedlings can be visually identified because of their glossy leaf surface which is different from the dull surface of wild-type seedlings. The Gl2 locus was isolated by transposon tagging. Seven unstable mutations, gl2-m2 to gl2-m8, were induced in a parental strain carrying an active transposable Activator (Ac) element in the unstable wx-m7 allele. Genetic tests on the gl2-m2 allele indicated that it was not caused by the Ac element but by the insertion of the transposable element Enhancer/Suppressor-Mutator (En/Spm). A Sa/l restriction fragment segregating with the mutant phenotype was identified, by Southern analysis, using sequences from the En/Spm element as a probe. Part of the fragment was cloned and was shown to carry part of the unstable gl2-m2 allele. These gl2 sequences were used to identify a genomic fragment carrying the wild-type allele and to isolate its corresponding cDNA sequence. The predicted Glossy2 protein consists of 426 amino acids. No similar amino acid sequence was found in protein data banks and the biochemical function of the Gl2 gene product is still unknown. The wild-type Gl2 transcript is found predominantly in juvenile leaves. The transcript level in the leaves of seedlings homozygous for a stable recessive gl2-ref allele is hardly detectable.

Alleles↗

PubMed2001

The selector gene cut represses a neural cell fate that is specified independently of the Achaete-Scute-Complex and atonal.

The peripheral nervous system (PNS) of Drosophila offers a powerful system to precisely identify individual cells and dissect their genetic pathways of development. The mode of specification of a subset of larval PNS cells, the multiple dendritic (md) neurons (or type II neurons), is complex and still poorly understood. Within the dorsal thoracic and abdominal segments, two md neurons, dbd and dda1, apparently require the proneural gene amos but not atonal (ato) or Achaete-Scute-Complex (ASC) genes. ASC normally acts via the neural selector gene cut to specify appropriate sensory organ identities. Here, we show that dbd- and dda1-type differentiation is suppressed by cut in dorsal ASC-dependent md neurons. Thus, cut is not only required to promote an ASC-dependent mode of differentiation, but also represses an ASC- and ato-independent fate that leads to dbd and dda1 differentiation.

Alleles↗

PubMed1988

Genetic interactions of modifier genes and modifiable alleles in Drosophila melanogaster.

We have examined the effects of mutations in the six allele-specific modifier genes su(Hw), e(we), su(f), su(s), su(wa), and su(pr) on the expression of 18 modifiable alleles, situated at 11 loci. Ten of the modifiable alleles are associated with insertions of the gypsy retrotransposon and the others include alleles associated with insertions of copia and 412. We tested or retested 90 of the 108 possible combinations and examined the expression of modifiable alleles in flies mutant for pairs of modifier genes in various heterozygous and homozygous configurations. Our principal findings are: (1) a screen of 40,000 mutagenized X chromosomes yielded three new mutations in known modifier genes, but revealed no new modifier genes; (2) the modification effects of different mutations in a given modifier gene were qualitatively similar; (3) each of the six modifiers suppressed some modifiable alleles, enhanced others, and had no noticeable effect on still others; (4) the modifier genes could be placed in four classes, according to their effects on the gypsy-insertion alleles; and (5) the effects of mutations in different modifier genes combined additively. Implications of these results for models of modifier gene action are discussed.

Alleles↗

PubMed2005

Pax3 functions at a nodal point in melanocyte stem cell differentiation.

Most stem cells are not totipotent. Instead, they are partially committed but remain undifferentiated. Upon appropriate stimulation they are capable of regenerating mature cell types. Little is known about the genetic programmes that maintain the undifferentiated phenotype of lineage-restricted stem cells. Here we describe the molecular details of a nodal point in adult melanocyte stem cell differentiation in which Pax3 simultaneously functions to initiate a melanogenic cascade while acting downstream to prevent terminal differentiation. Pax3 activates expression of Mitf, a transcription factor critical for melanogenesis, while at the same time it competes with Mitf for occupancy of an enhancer required for expression of dopachrome tautomerase, an enzyme that functions in melanin synthesis. Pax3-expressing melanoblasts are thus committed but undifferentiated until Pax3-mediated repression is relieved by activated beta-catenin. Thus, a stem cell transcription factor can both determine cell fate and simultaneously maintain an undifferentiated state, leaving a cell poised to differentiate in response to external stimuli.

Aging↗

PubMed1999

Transcriptional targeting of retroviral vectors to the erythroblastic progeny of transduced hematopoietic stem cells.

Targeted expression to specific tissues or cell lineages is a necessary feature of a gene therapy vector for many clinical applications, such as correction of hemoglobinopathies or thalassemias by transplantation of genetically modified hematopoietic stem cells. We developed retroviral vectors in which the constitutive viral enhancer in the U3 region of the 3' LTR is replaced by an autoregulatory enhancer of the erythroid-specific GATA-1 transcription factor gene. The replaced enhancer is propagated to the 5' LTR upon integration into the target cell genome. The modified vectors were used to transduce human hematopoietic cell lines, cord blood-derived CD34(+) stem/progenitor cells, and murine bone marrow repopulating stem cells. The expression of appropriate reporter genes (triangle upLNGFR, EGFP) was analyzed in the differentiated progeny of transduced stem cells in vitro, in liquid culture as well as in clonogenic assay, and in vivo, after bone marrow transplantation in lethally irradiated mice. The GATA-1 autoregulatory enhancer effectively restricts the expression of the LTR-driven proviral transcription unit to the erythroblastic progeny of both human progenitors and mouse-repopulating stem cells. Packaging of viral particles, integration into the target genome, and stability of the integrated provirus are not affected by the LTR modification. Enhancer replacement is therefore an effective strategy to target expression of a retroviral transgene to a specific progeny of transduced hematopoietic stem cells.

Animals↗

PubMed2004

Protein-only transmission of three yeast prion strains.

Key questions regarding the molecular nature of prions are how different prion strains can be propagated by the same protein and whether they are only protein. Here we demonstrate the protein-only nature of prion strains in a yeast model, the [PSI] genetic element that enhances the read-through of nonsense mutations in the yeast Saccharomyces cerevisiae. Infectious fibrous aggregates containing a Sup35 prion-determining amino-terminal fragment labelled with green fluorescent protein were purified from yeast harbouring distinctive prion strains. Using the infectious aggregates as 'seeds', elongated fibres were generated in vitro from the bacterially expressed labelled prion protein. De novo generation of strain-specific [PSI] infectivity was demonstrated by introducing sheared fibres into uninfected yeast hosts. The cross-sectional morphology of the elongated fibres generated in vitro was indistinguishable from that of the short yeast seeds, as visualized by electron microscopy. Electron diffraction of the long fibres showed the 4.7 A spacing characteristic of the cross-beta structure of amyloids. The fact that the amyloid fibres nucleated in vitro propagate the strain-specific infectivity of the yeast seeds implies that the heritable information of distinct prion strains must be encoded by different, self-propagating cross-beta folding patterns of the same prion protein.

Amyloid↗

PubMed2000

The Drosophila cystoblast differentiation factor, benign gonial cell neoplasm, is related to DExH-box proteins and interacts genetically with bag-of-marbles.

Selection of asymmetric cell fates can involve both intrinsic and extrinsic factors. Previously we have identified the bag-of-marbles (bam) gene as an intrinsic factor for cystoblast fate in Drosophila germline cells and shown that it requires active product from the benign gonial cell neoplasm (bgcn) gene. Here we present the cloning and characterization of bgcn. The predicted Bgcn protein is related to the DExH-box family of RNA-dependent helicases but lacks critical residues for ATPase and helicase functions. Expression of the bgcn gene is extremely limited in ovaries but, significantly, bgcn mRNA is expressed in a very limited number of germline cells, including the stem cells. Also, mutations in bgcn dominantly enhance a bam mutant phenotype, further corroborating the interdependence of these two genes' functions. On the basis of known functions of DExH-box proteins, we propose that Bgcn and Bam may be involved in regulating translational events that are necessary for activation of the cystoblast differentiation program.

Alleles↗

PubMed2002

A lin-45 raf enhancer screen identifies eor-1, eor-2 and unusual alleles of Ras pathway genes in Caenorhabditis elegans.

In Caenorhabditis elegans, the Ras/Raf/MEK/ERK signal transduction pathway controls multiple processes including excretory system development, P12 fate specification, and vulval cell fate specification. To identify positive regulators of Ras signaling, we conducted a genetic screen for mutations that enhance the excretory system and egg-laying defects of hypomorphic lin-45 raf mutants. This screen identified unusual alleles of several known Ras pathway genes, including a mutation removing the second SH3 domain of the sem-5/Grb2 adaptor, a temperature-sensitive mutation in the helical hairpin of let-341/Sos, a gain-of-function mutation affecting a potential phosphorylation site of the lin-1 Ets domain transcription factor, a dominant-negative allele of ksr-1, and hypomorphic alleles of sur-6/PP2A-B, sur-2/Mediator, and lin-25. In addition, this screen identified multiple alleles of two newly identified genes, eor-1 and eor-2, that play a relatively weak role in vulval fate specification but positively regulate Ras signaling during excretory system development and P12 fate specification. The spectrum of identified mutations argues strongly for the specificity of the enhancer screen and for a close involvement of eor-1 and eor-2 in Ras signaling.

Alleles↗

PubMed2006

Zinc-finger genes Fez and Fez-like function in the establishment of diencephalon subdivisions.

Fez and Fez-like (Fezl) are zinc-finger genes that encode transcriptional repressors expressed in overlapping domains of the forebrain. By generating Fez;Fezl-deficient mice we found that a redundant function of Fez and Fezl is required for the formation of diencephalon subdivisions. The caudal forebrain can be divided into three transverse subdivisions: prethalamus (also called ventral thalamus), thalamus (dorsal thalamus) and pretectum. Fez;Fezl-deficient mice showed a complete loss of prethalamus and a strong reduction of the thalamus at late gestation periods. Genetic marker analyses revealed that during early diencephalon patterning in Fez;Fezl-deficient mice, the rostral diencephalon (prospective prethalamus) did not form and the caudal diencephalon (prospective thalamus and pretectum) expanded rostrally. Fez;Fezl-deficient mice also displayed defects in the formation of the zona limitans intrathalamica (ZLI), which is located on the boundary between the prethalamus and thalamus. Fez and Fezl are expressed in the region rostral to the rostral limit of Irx1 expression, which marks the prospective position of the ZLI. Transgene-mediated misexpression of Fezl or Fez caudal to the ZLI repressed the caudal diencephalon fate and affected the formation of the Shh-expressing ZLI. These data indicate that Fez and Fezl repress the caudal diencephalon fate in the rostral diencephalon, and ZLI formation probably depends on Fez/Fezl-mediated formation of diencephalon subdivisions.

Animals↗

PubMed1997

Genetic enhancers of sem-5 define components of the gonad-independent guidance mechanism controlling sex myoblast migration in Caenorhabditis elegans hermaphrodites.

The migrations of the sex myoblasts in Caenorhabditis elegans hermaphrodites involve two guidance mechanism: a gonad-dependent attraction that confers precise positioning of the sex myoblasts and a gonad-independent mechanism that is sufficient for coarse positioning in the absence of the gonad (Thomas et al., 1990). Here we show that mutations in unc-53, unc-71, and unc-73 disrupt sex myoblast positioning in the absence of the gonad, while they do not affect positioning in the presence of the gonad. Thus, mutations in these genes appear to compromise the gonad-independent mechanism without affecting motility or the gonad-dependent attraction. Mutations in sem-5 confer dramatic sex myoblast positioning defects in double mutant combinations with unc-53, unc-71, or unc-73 mutations, even in the presence of the gonad. This suggests that sem-5 is required for the gonad-dependent attractive mechanism. Mutations in let-60 ras and let-341 also confer sex myoblast migration defects in an unc-53 background, implicating these genes in gonad-dependent positioning as well.

Animals↗