Extensive Genetic Screening Research Articles

Modulation of 1O2‐mediated retrograde signaling by the PLEIOTROPIC RESPONSE LOCUS 1 (PRL1) protein, a central integrator of stress and energy signaling

Shortly after the release of singlet oxygen ((1)O(2)) in chloroplasts, changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. Extensive genetic screens aimed at identifying constituents involved in (1)O(2)-mediated plastid-to-nucleus signaling have failed to identify extraplastidic signaling components. This finding suggests that (1)O(2)-mediated signals are not translocated to the nucleus via a single linear pathway, but rather through a signaling network that is difficult to block by single mutations. The complexity of this signaling network has been tackled by mutagenizing a transgenic flu line expressing the luciferase reporter gene under the control of the promoter of a (1)O(2)-responsive AAA-ATPase gene (At3g28580) and isolating second site mutants that constitutively express the reporter gene at a high level. One of the mutants was shown by map-based cloning and sequencing to contain a single amino acid change in the PLEIOTROPIC RESPONSE LOCUS 1 (PRL1) protein. PRL1 suppresses the expression of AAA-ATPase and other (1)O(2)-responsive genes. PRL1 seems to play a major role in modulating responses of plants to environmental changes by interconnecting (1)O(2)-mediated retrograde signaling with other signaling pathways.

BCR-induced cell death of B cells from CD22 deficient mice is mediated by a novel ssRNA-directed endonuclease (136.33)

Abstract B cells from CD22 deficient mice backcrossed onto a C57BL/6 genetic background (B6 CD22-/-) undergo B cell antigen receptor (BCR)-induced apoptosis. By contrast, B cells from parental inbred CD22-/- mice on a mixed B6x129 genetic background (B6/129 CD22-/-) survive and proliferate normally following BCR ligation. Through an extensive reverse genetic screen, a single locus was identified as being responsible for the proliferation defect of B6 CD22-/- B cells. In B6 CD22-/- mice this locus was homozygous for B6 germline DNA (locusB6), and in B6/129 CD22-/- mice this region was homozygous for 129 germline DNA (locus129). In locusB6 CD22-/- B cells, a novel single-stranded RNA (ssRNA)-directed endonuclease was specifically overexpressed compared to locus129 CD22-/- B cells, suggesting a direct role in BCR-induced cell death. Targeted disruption of the endonuclease gene in locusB6 CD22-/- mice restored BCR-mediated B cell survival and proliferation to normal levels. These results identify a novel ssRNA-directed endonuclease as a potent inhibitor of B cell survival when expressed following antigen receptor stimulation. This research was supported by NIH grant R01CA096547.

Mutations of genes in synthesis of the carotenoid precursors of ABA lead to pre‐harvest sprouting and photo‐oxidation in rice

Pre-harvest sprouting (PHS) or vivipary in cereals is an important agronomic trait that results in significant economic loss. A considerable number of mutations that cause PHS have been identified in several species. However, relatively few viviparous mutants in rice (Oryza sativa L.) have been reported. To explore the mechanism of PHS in rice, we carried out an extensive genetic screening and identified 12 PHS mutants (phs). Based on their phenotypes, these phs mutants were classified into three groups. Here we characterize in detail one of these groups, which contains mutations in genes encoding major enzymes of the carotenoid biosynthesis pathway, including phytoene desaturase (OsPDS), ζ-carotene desaturase (OsZDS), carotenoid isomerase (OsCRTISO) and lycopene β-cyclase (β-OsLCY), which are essential for the biosynthesis of carotenoid precursors of ABA. As expected, the amount of ABA was reduced in all four phs mutants compared with that in the wild type. Chlorophyll fluorescence analysis revealed the occurrence of photoinhibition in the photosystem and decreased capacity for eliminating excess energy by thermal dissipation. The greatly increased activities of reactive oxygen species (ROS) scavenging enzymes, and reduced photosystem (PS) II core proteins CP43, CP47 and D1 in leaves of the Oscrtiso/phs3-1 mutant and OsLCY RNAi transgenic rice indicated that photo-oxidative damage occurred in PS II, consistent with the accumulation of ROS in these plants. These results suggest that the impairment of carotenoid biosynthesis causes photo-oxidation and ABA-deficiency phenotypes, of which the latter is a major factor controlling the PHS trait in rice.

Iron Control of Erythropoiesis: A Novel Pathway in Which Aconitase Enzymes Couple Metabolic Flux with Cellular Development.

Erythroid iron deficiency, whether due to dimished body stores or histiocytic retention, diminishes marrow responsiveness to erythropoietin (Epo). Conversely, intravenous iron infusion augments marrow responsiveness to Epo, even in anemia patients with adequate pre-existing iron stores. Iron regulation of Epo-driven erythropoiesis affects proliferation and differentiation of early progenitors, prior to the commitment to heme production. Thus, while iron is essential for all cells, erythroid precursors manifest an exquisite sensitivity to iron deficiency, most likely as a rationing mechanism to protect other, more vital iron-dependent functions. Using primary human hematopoietic cultures with defined levels of transferrin saturation, we have confirmed the existence of a critical threshold of iron deprivation, at which erythroid progenitors display proliferative and maturation blockade while granulopoiesis and megakaryopoiesis remain unaffected. Extensive pharmacologic and genetic screening for components of this erythroid iron response pathway have identified the iron-sulfur cluster containing aconitase enzymes as a critical signaling node. Mitochondrial and cytosolic aconitase (mAcon & cAcon) interconvert citrate and isocitrate as a key step in the Krebs cycle. Firstly, treatment of iron deprived erythroid cultures with exogenous isocitrate, but not citrate, completely restored differentiation, as judged by glycophorin A (GPA) expression and hemoglobinization. By contrast, both citrate and isocitrate enhanced erythroid differentiation under iron replete conditions. Secondly, treatment of iron replete erythroid cultures with a specific aconitase inhibitor, fluorocitrate, induced a lineage-selective maturation blockade identical to that seen with iron deprivation. Thirdly, enzymography showed erythroid-lineage specific inactivation of both cAcon and mAcon in response to iron deprivation; immunoblotting showed no change mAcon protein levels as a function of either lineage or iron status. Fourthly, a retroviral genetic screen identified HBLD2, an iron-sulfur cluster assembly factor, as a protein whose overexpression reversed the erythroid maturation blockade associated with iron deprivation. Enzymography confirmed that overexpression of HBLD2 enhanced both cAcon and mAcon activities. Fifthly, administration to wild type, iron replete C57BL/6 mice of isocitrate at 200 mg/kg/day for 5 days caused a significant increase in peripheral red cell hemoglobinization, reflected by MCHC and Hb levels. Taken together, these results identify isocitrate as a positive regulator of Epo-mediated erythroid differentiation. In Epo-independent CD34+ cell culture systems, isocitrate did not enhance erythropoiesis. Therefore aconitase enzymes serve as a critical nexus in iron and Epo regulation of erythropoiesis, integrating cellular metabolism with developmental programming. The unique sensitivity of the erythroid lineage to iron deprivation appears to derive from a cellular milieu effect on the aconitase enzymes, promoting their inactivation under iron deprivation. Notably, IRP repression of mAcon translation does not contribute to the erythroid iron deprivation response pathway. This pathway may have relevance for future clinical approaches to Epo-refractory chronic anemias and polycythemia vera.

Development of a Genetic Tool Based on BCR-ABL Transgenic Drosophila Melanogaster for Identification of Genes Involved in CML Progression and Drug Testing.

Although the role of Bcr-Abl in the pathogenesis of chronic myeloid leukaemia is well established, the molecular mechanisms by which it triggers cellular transformation remains still partially unknown. In addition, the mechanisms responsible for CML progression and the molecules interacting with Bcr-Abl are largely unknown. and many of. In this study, we have developed a novel approach for genetic analysis investigations to identify a number of candidate genes and pathways involved in disease progression and imatinib resistance by using a model of the Drosophila melanogaster. We generated two different stable transgenic fly lines expressing both human p210Bcr-Abl forms (either w.t. or the mutated form T315I) in a tissue specific manner. We have observed that the activation of BCR-ABL led to a particular phenotype in the fly eyes. Transgenic flies will be phenotipically and genotipically characterized carefully by analyzing the eye development. We conducted an extensive genetic screening using both Drosphila transgenic lines overexpressing human Bcr-Abl forms (Bcr-Abl/w.t. and Bcr-Abl/T315I) by making use of P-elements. This technique offers the possibility to randomly insert P-elements into the genome, thus disrupting genomic loci either in correspondence of coding sequences or regulatory elements and altering the gene function. The heterogeneous mutagenized fly population was carefully screened on the basis of the flies phenotype. The first group which we have selected was represented by flies displaying a phenotype even more aggressive than the parental transgenic fly. This kind of flies harbour mutated genes encoding for proteins which enhance the activity of Bcr-Abl, thus being most likely involved in disease progression; A second group was represented by flies displaying a wild-type phenotype. In the latter case the phenotype reversion most likely due to a mutation occurred at a level of a gene encoding for a protein functioning as Bcr-Abl negative regulator. All the data obtained with the use of fly model were confirmed in both cell lines and in primary cells via the overexpression and/or silencing of the genes identified with the Drosophila genetic-screening. Finally we have established and validated a novel tool for drug testing based on the examination of the eye phenotype induced by Bcr-Abl, which can be reverted by the drugs producing a complete block of the kinase activity. With this method we will be able to screen drug libraries to identify molecules able to silence Bcr-AblT315I tyrosine kinase activity. That can be easily accomplished by feeding flies with food previously mixed with the different drug molecules. Molecules showing a good inhibitory activity can be quickly identified because their /ability to revert the abnormal eye phenotype displayed by the transgenic flies. In conclusion, by using a model of the Drosophila melanogaster we have developed a system to identify novel genetic pathways which regulate the development and the progression of CML and we have shown that this novel system can be also used to evaluate the drugs affecting Bcr-Abl regulatory pathway. Moreover, this system does not require a priori knowledge of the function of the genes involved in the disease.

Apports de la spectroscopie par résonance magnétique nucléaire des fluides dans l’étude de maladies métaboliques et neurodégénératives

In vitro Nuclear Magnetic Resonance (NMR) spectroscopy is a validated biochemical tool for metabolic analyses of human body fluids and diagnosis of inborn errors of metabolism in children and adults. The technique is of special interest because it requires minimal sample preparation, it can detect simultaneously compounds of different nature and it offers structural information on the metabolites present in body fluids. In the last decade, in vitro NMR spectroscopy contributed to the identification of new inborn errors of metabolism, some of which are amenable to therapeutic intervention. Standardized analyses of body fluids, especially cerebrospinal fluid, are therefore indicated in patients affected with neurodegenerative disorders of unknown etiologies, for which extensive metabolic and genetic screening failed to identify the primary defect. In addition, the use of multivariate statistical analyses allows the comparison of the whole metabolic profile between patients with a given neurodegenerative disorder and controls. This so-called metabonomic approach yields promises for the discovery of biomarkers that can help detecting disease onset and progression, as well as evaluating therapeutic efficacy. Finally, the combination of in vitro NMR spectroscopy with genetic analytical tools may constitute a successful pathophysiological approach to investigate neurological disorders of unknown etiology.

FBXW7/hCDC4 Is a General Tumor Suppressor in Human Cancer

The ubiquitin-proteasome system is a major regulatory pathway of protein degradation and plays an important role in cellular division. Fbxw7 (or hCdc4), a member of the F-box family of proteins, which are substrate recognition components of the multisubunit ubiquitin ligase SCF (Skp1-Cdc53/Cullin-F-box-protein), has been shown to mediate the ubiquitin-dependent proteolysis of several oncoproteins including cyclin E1, c-Myc, c-Jun, and Notch. The oncogenic potential of Fbxw7 substrates, frequent allelic loss in human cancers, and demonstration that mutation of FBXW7 cooperates with p53 in mouse tumorigenesis have suggested that Fbxw7 could function as a tumor suppressor in human cancer. Here, we carry out an extensive genetic screen of primary tumors to evaluate the role of FBXW7 as a tumor suppressor in human tumorigenesis. Our results indicate that FBXW7 is inactivated by mutation in diverse human cancer types with an overall mutation frequency of approximately 6%. The highest mutation frequencies were found in tumors of the bile duct (cholangiocarcinomas, 35%), blood (T-cell acute lymphocytic leukemia, 31%), endometrium (9%), colon (9%), and stomach (6%). Approximately 43% of all mutations occur at two mutational "hotspots," which alter Arg residues (Arg465 and Arg479) that are critical for substrate recognition. Furthermore, we show that Fbxw7Arg465 hotspot mutant can abrogate wild-type Fbxw7 function through a dominant negative mechanism. Our study is the first comprehensive screen of FBXW7 mutations in various human malignancies and shows that FBXW7 is a general tumor suppressor in human cancer.

Pristionchus pacificus: a well‐rounded nematode

Nematodes pervade Earth's biosphere and occupy innumerable ecological niches. The role of Caenorhabditis elegans as a model for developmental processes has encouraged us to cultivate a second nematode, Pristionchus pacificus, as a comparative counterpoint to address questions in development, behavior and ecology in nematode evolution. We hope that this endeavor, now more than a decade underway, will allow us to project findings onto other comparative models for biological processes. To this end, our laboratory has made an extensive genetic map and mutant screens to understand changes in developmental programs. Recently, we have been capitalizing on the whole genome sequence of P. pacificus to describe more thoroughly the molecular basis for these changes, as well as to better integrate our molecular knowledge with the biodiversity of Pristionchus species.

Repression of Cell-Cell Fusion by Components of the C. elegans Vacuolar ATPase Complex

Cell-cell fusion initiates fertilization, sculpts tissues during animal development, reprograms stem cells to new differentiated states, and may be a key step in cancer progression. While cell fusion is tightly regulated, the mechanisms that limit fusion to appropriate partners are unknown. Here, we report that the fus-1 gene is essential to repress fusion of epidermal cells in C. elegans: in severe fus-1 mutants, all epidermal cells, except the lateral seam cells, inappropriately fuse into a single large syncytium. This hyperfusion requires EFF-1, an integral membrane protein essential for fusion of epidermal cells into discrete syncytia. FUS-1 is localized to the apical plasma membrane in all epidermal cells potentiated to undergo fusion, whereas it is virtually undetectable in nonfusing seam cells. fus-1 encodes the e subunit of the vacuolar H(+)-ATPase (V-ATPase), and loss of other V-ATPase subunits also causes widespread hyperfusion. These findings raise the possibility of manipulating cell fusion by altering V-ATPase activity.

Bypassing Cellular Senescence by Ezh2.

In this study mouse embryonic fibroblasts (MEFs) and hematopoietic stem cells (HSCs) were used to identify genes involved in the process of cellular aging. MEFs are able to undergo a distinct number of population doublings, after which they enter a state of irreversible growth arrest termed replicative senescence. Previous studies of others and us have shown that aging is regulated by an intrinsic genetic program. In order to identify candidate genes involved in senescence we assessed gene expression profiles of proliferating and senescent MEFs. Interestingly, a significant number of genes that was higher expressed in proliferating vs. senescent MEFs, are involved in epigenetic regulation of gene transcription by modulating histone methylation and deacetylation. The top candidate gene was Enhancer of zeste homolog 2 (Ezh2), a Polycomb group protein (PcG), involved in histone methylation and deacetylation and known to function as a negative regulator of gene transcription. It has been reported that Ezh2 is essential during development, since Ezh2-deficient mice die early during embryonic development. Ezh2 expression was rapidly downregulated during senescence in primary MEFs, but was readily detectable in spontaneously transformed cells. Retroviral overexpression of Ezh2 in primary MEFs resulted in bypassing the senescence program. In addition, overexpression of Ezh2 in spontaneously transformed MEFs increased proliferation rates. We found that Ezh2 is highly expressed in purified Lin−Sca-1+c-kit+ (LSK) HSCs. In agreement with MEF studies, terminal differentiation of stem cells in vitro resulted in rapid downregulation of Ezh2 expression. Furthermore, overexpression of Ezh2 in purified HSCs results in increased proliferation rate in vitro. Moreover, HSCs transduced with Ezh2 were enriched for CFU-GM activity. Ongoing studies will address whether overexpression of Ezh2 in stem cells will affect their self-renewal potential in vivo. In an extensive genetic screen we identified genes that correlate with Ezh2 expression in stem cells, potential targets or partners of Ezh2, among others Top2a, MKi67, Pcna, and Eed. These data were deposited in an on-line resource (www.WebQTL.org), to uncover genetic networks that are involved in regulating the process of aging.

Nasonia

What is Nasonia? A small (2–3mm) parasitoid wasp that oviposits in the pupae of flies (but not those of Drosophila!). This hymenopteran occurs naturally in bird nests and at carcasses. Three species are known: the cosmopolitan N. vitripennis and two endemic North American species, N. giraulti and N. longicornis. Nasonia is extremely easy to culture on commercially available host pupae, it has a short generation time (15 days at 25°C) and can be maintained in diapause for over one year. What is so special about Nasonia?Nasonia has haplodiploid reproduction; haploid males develop from unfertilized eggs and diploid females from fertilized eggs. Thus, all recessive traits are expressed directly in the male: this is the case for X chromosome genes in Drosophila, but with Nasonia it is true of the entire genome. An extensive forward genetic screen for developmental mutants has already been accomplished and the identification of candidate genes is under way. Unlike the honey bee and many other hymenopterans, Nasonia has no single locus complementary sex determination, which allows fully homozygous strains to be kept without inbreeding problems. Another major advantage of Nasonia is that the three species, which exhibit considerable morphological and behavioral differences, can produce viable and fertile hybrids in the laboratory. The genetic basis for these differences can thus be easily identified through QTL analysis. Nasonia is also very favorable for cell biology, with its very clear yolk and slow early development which allow easy imaging. A useful species for studying the evolution of development then? Yes. General patterns in development and evolution can only come from comparison of multiple organisms. Nasonia has a long germband mode of development, which facilitates comparison of expression patterns with Drosophila; in contrast the beetle Tribolium, also much used for studies of the evolution of development, exhibits short germband development. Transgenic and RNAi tools are also being developed for Nasonia. With respect to sex determination, it will be exciting to figure out how ploidy differences lead to determination of the two sexes. What aspects of evolutionary genetics might Nasonia illuminate?Nasonia is extensively used to study the evolution of sex ratios and speciation. Females vary the sex of their offspring by controlling whether eggs are fertilized. A number of non-Mendelian elements, including the ‘selfish’ paternal sex-ratio chromosome and cytoplasmic sex-ratio distorters illustrate genetic conflict over sex determination. Nasonia is one of the primary organisms used to investigate the evolution and mechanisms of action of Wolbachia, an intracellular bacterium which infects many arthropods. Finally, exciting work is being done, using the three cross-fertile species, on the genetic basis of hybrid incompatibilities and interspecies differences in morphology and behaviour (wingsize and courtship, for example). This takes advantage of exchange of genes or genomic regions between species and the construction of introgression lines in which a gene for a particular trait from one species is bred into another species' genetic background. Are there genomic tools available for Nasonia? Efforts are already underway to develop genetic and genomic tools for Nasonia. Visible genetic markers are known from all five chromosomes. Dense linkage maps containing RAPD, AFLP and microsatellite markers are available with a resolution of approximately 1cM, making possible whole genome screens for mutations and fine-scale mapping of genes. The total genome size is ∼335 Mb and 1cM corresponds to ∼100 Kb of genomic DNA. An embryonic cDNA library and a BAC library funded by NSF have been constructed, and the first attempts at positional cloning are underway, focusing on a major gene for wingsize. Where can I find out more?

Age-related macular degeneration: a new viewpoint.

Age-related macular degeneration (AMD) is a major cause of blindness in the United States. AMD can be categorized into an atrophic (dry) form and a neovascular (wet, exudative) form. The atrophic form involves alterations of pigment distribution, loss of RPE cells and photoreceptors and diminished retinal function due to an overall atrophy of the cells. The neovascular AMD involves proliferation of abnormal choroidal vessels, which penetrate the Bruch's membrane and RPE layer into the subretinal space, thereby forming extensive clots and/or scars. Both environmental and genetic factors are suspected to play a role in AMD. Despite extensive genetic screening of candidate genes only two associations have been identified with AMD (Adenosine triphosphate (ATP)-binding cassette rim (ABCR) protein and apolipoprotein E gene-ApoE). The ABCR protein is retinal specific and accounts for only 3% of AMD cases. ApoE is not specific to the retina, and has been more intriguingly associated with Alzheimer's, another disease of age. The most consistent major risk factor in AMD is age. Our studies on the ACE gene show an association of protection with an Alu element insert, which might be affecting the level of the ACE gene. The ApoE 4 allele and the ACE Alu+/+ genotype have both been shown to be a risk for Alzheimer's and protective for AMD. Given these recent genetic associations, we should examine possible common pathways in diseases of age and their interaction with human genetic polymorphisms.

Role of parkin mutations in 111 community-based patients with early-onset parkinsonism.

Early-onset parkinsonism is frequently reported in connection with mutations in the parkin gene. In this study, we present the results of extensive genetic screening for parkin mutations in 111 community-derived early-onset parkinsonism patients (age of onset <50 years) from Germany with an overall mutation rate of 9.0%. Gene dosage alterations represented 67% of the mutations found, underlining the importance of quantitative analyses of parkin. In summary, parkin mutations accounted for a low but significant percentage of early-onset parkinsonism patients in a community-derived sample.

Transcription activation by a variety of AraC/XylS family activators does not depend on the class II-specific activation determinant in the N-terminal domain of the RNA polymerase alpha subunit.

The N-terminal domain of the RNA polymerase alpha subunit (alpha-NTD) was tested for a role in transcription activation by a variety of AraC/XylS family members. Based on substitutions at residues 162 to 165 and an extensive genetic screen we conclude that alpha-NTD is not an activation target for these activators.