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GIGANTEA acts in blue light signaling and has biochemically separable roles in circadian clock and flowering time regulation.

Circadian clocks are widespread in nature. In higher plants, they confer a selective advantage, providing information regarding not only time of day but also time of year. Forward genetic screens in Arabidopsis (Arabidopsis thaliana) have led to the identification of many clock components, but the functions of most of these genes remain obscure. To identify both new constituents of the circadian clock and new alleles of known clock-associated genes, we performed a mutant screen. Using a clock-regulated luciferase reporter, we isolated new alleles of ZEITLUPE, LATE ELONGATED HYPOCOTYL, and GIGANTEA (GI). GI has previously been reported to function in red light signaling, central clock function, and flowering time regulation. Characterization of this and other GI alleles has helped us to further define GI function in the circadian system. We found that GI acts in photomorphogenic and circadian blue light signaling pathways and is differentially required for clock function in constant red versus blue light. Gene expression and epistasis analyses show that TIMING OF CHLOROPHYLL A/B BINDING PROTEIN1 (TOC1) expression is not solely dependent upon GI and that GI expression is only indirectly affected by TOC1, suggesting that GI acts both in series with and in parallel to TOC1 within the central circadian oscillator. Finally, we found that the GI-dependent promotion of CONSTANS expression and flowering is intact in a gi mutant with altered circadian regulation. Thus GI function in the regulation of a clock output can be biochemically separated from its role within the circadian clock.

Alleles↗

Screening of the rice viviparous mutants generated by endogenous retrotransposon Tos17 insertion. Tagging of a zeaxanthin epoxidase gene and a novel ostatc gene.

The rice (Oryza sativa) retrotransposon Tos17 is one of a few active retrotransposons in plants and its transposition is activated by tissue culture. Here, we present the characterization of viviparous mutants of rice induced by tissue culture to demonstrate the feasibility of the use of retrotransposon Tos17 as an endogenous insertional mutagen and cloning of the tagged gene for forward genetics in unraveling the gene function. Two mutants were shown to be caused by the insertion of Tos17. Osaba1, a strong viviparous mutant with wilty phenotype, displayed low abscisic acid level and almost no further increase in its levels upon drought. The mutant is shown to be impaired in the epoxidation of zeaxanthin. On the other hand, Ostatc, a mutant with weak phenotype, exhibited the pale green phenotype and slight increase in abscisic acid levels upon drought. Deduced amino acids of the causative genes of Osaba1 and Ostatc manifested a significantly high homology with zeaxanthin epoxidase isolated from other plant species and with bacterial Sec-independent translocase TATC protein, respectively. This is the first example of transposon tagging in rice.

Abscisic Acid↗

The Arabidopsis SPIKE1 gene is required for normal cell shape control and tissue development.

Regulated growth and cell shape control are fundamentally important to the function of plant cells, tissues, and organs. The signal transduction cascades that control localized growth and cell shape, however, are not known. To better understand the relationship between cytoskeletal organization, organelle positioning, and regulated vesicle transport, we conducted a forward genetic screen to identify genes that regulate cytoskeletal organization in plants. Because of the distinct requirements for microtubules and actin filaments during leaf trichome development, a trichome-based morphology screen is an efficient approach to identify genes that affect cytoplasmic organization. The seedling lethal spike1 mutant was identified based on trichome, cotyledon, and leaf-shape defects. The predicted SPIKE1 protein shares amino acid identity with a large family of adapter proteins present in humans, flies, and worms that integrate extracellular signals with cytoskeletal reorganization. Both the trichome phenotype and immunolocalization data suggest that SPIKE1 also is involved in cytoskeletal reorganization. The assembly of laterally clustered foci of microtubules and polarized growth are early events in cotyledon development, and both processes are misregulated in spike1 epidermal cells.

Amino Acid Sequence↗

Addiction and its brain science.

AIMS: To illustrate how modern neurobiological approaches will help to identify the neurocircuits and genes involved in addictive behavior. BACKGROUND: The current disorder concept of addiction includes neurobiological foundations and neurobiological research assuming irreversible molecular and structural changes within the brain dopamine reinforcement system, constituting the 'molecular and structural switch' from controlled drug intake to compulsive drug abuse. However, those irreversible changes have not so far been identified and it is suggested that in addition to the mesolimbic dopamine system, other brain systems including the mesocortical and nigrostriatal pathways as well as their non-dopaminergic feedback-loops might be involved in addictive behavior. NEUROBIOLOGICAL APPROACH: A three-step neurobiological approach is described that allows in a first step via novel animal models and imaging techniques to identify the neuroanatomical sites mediating voluntary drug intake, reinstatement of drug-seeking behavior, relapse, loss of control and drug intake despite negative consequences. In a subsequent step, forward genetic approaches including quantitative trait loci (QTL)-analysis and gene expression profiling are helpful in identifying so-called candidate genes. In a final step, conditional animal mutants and selective pharmacological tools are used to functionally validate candidate genes. Following this validation process, the transfer to the human situation has to be made and candidate genes have to be verified further in well-phenotyped cohorts of addicted patients. CONCLUSION: This three-step neurobiological approach, that must involve an interdisciplinary team including experimental psychologists, geneticists, molecular biologists and finally clinical addiction researchers, will allow us to understand where and how the addicted brain goes awry.

Animals↗

An alpha-(1,4)-amylase is essential for alpha-(1,3)-glucan production and virulence in Histoplasma capsulatum.

Histoplasma capsulatum is a dimorphic fungus that causes respiratory and systemic disease and is capable of surviving and replicating within macrophages. The virulence of Histoplasma has been linked to cell wall alpha-(1,3)-glucan; however, the role of this polysaccharide during infection, its organization within the cell wall, and its synthesis and regulation remain poorly understood. To identify genes involved in the biosynthesis of alpha-(1,3)-glucan, we employed a forward genetics strategy to isolate physically marked mutants with reduced alpha-(1,3)-glucan. Insertional mutants were generated in a virulent strain of H. capsulatum by optimization of Agrobacterium tumefaciens-mediated transformation. Approximately 90% of these mutants possessed single insertions with no chromosomal rearrangements or deletions in the host genome. To confirm the role and specificity of identified candidate genes, we phenocopied the disrupted locus by either RNA interference or targeted gene deletion. Our findings indicate alpha-(1,3)-glucan production requires the function of the AMY1 gene product, a novel protein with homology to the alpha-amylase family of glycosyl hydrolases, and UGP1, a UTP-glucose-1-phosphate uridylyltransferase which synthesizes UDP-glucose monomers. Loss of AMY1 function attenuated the ability of Histoplasma to kill macrophages and to colonize murine lungs.

Agrobacterium tumefaciens↗

The symbiotic ion channel homolog DMI1 is localized in the nuclear membrane of Medicago truncatula roots.

Legumes utilize a common signaling pathway to form symbiotic associations both with rhizobial bacteria and arbuscular mycorrhizal fungi. The perception of microbial signals is believed to take place at the plasma membrane, activating a cascade that converges on the nucleus where transcriptional reprogramming facilitates the symbioses. Forward genetic strategies have identified genes in this signaling pathway including Medicago truncatula DMI1 (Doesn't Make Infections 1) that encodes a putative ion channel. Although the DMI1 homologs from Lotus japonicus, CASTOR and POLLUX, were recently reported to be localized in plastids, we report here that a functional DMI1::GFP fusion is localized to the nuclear envelope in M. truncatula roots when expressed both from a constitutive 35S promoter and from a native DMI1 promoter. Localization may be mediated in part by sequences located within the amino-terminus of DMI1. This region of DMI1 is required for symbiotic signal transduction, and its replacement with a bona fide plastid transit peptide from the glutamine synthetase 2 gene does not restore DMI1 function. These new data place DMI1 in the nuclear envelope in close proximity to the origin of Nod-factor-induced calcium spiking.

Cell Nucleus↗

ird1 is a Vps15 homologue important for antibacterial immune responses in Drosophila.

The immune response-deficient 1 (ird1) gene was identified in a forward genetic screen as a novel regulator for the activation of Imd NFkappaB immune signalling pathway in Drosophila. ird1 animals are also more susceptible to Escherichia coli and Micrococcus luteus bacterial infection. ird1 encodes the Drosophila homologue of the Vps15/p150 serine/threonine kinase that regulates a class III phosphoinositide 3-kinase and is necessary for phagosome maturation and starvation-induced autophagy in yeast and mammalian cells. To gain insight into the role of ird1 in the immune response, we examine how amino acid starvation affects the immune signalling pathways in Drosophila. Starvation, in the absence of infection, leads to expression of antimicrobial peptide (AMP) genes and this response is dependent on ird1 and the Imd immune signalling pathway. Starvation, in addition to bacterial infection, suppresses the AMP response in wild-type animals and reduces the ability to survive M. luteus infection. Our results suggest that starvation and innate immune signalling may be intimately linked processes.

Animals↗

TRP channels and lipids: from Drosophila to mammalian physiology.

The transient receptor potential (TRP) ion channel family was the last major ion channel family to be discovered. The prototypical member (dTRP) was identified by a forward genetic approach in Drosophila, where it represents the transduction channel in the photoreceptors, activated downstream of a Gq-coupled PLC. In the meantime 29 vertebrate TRP isoforms are recognized, distributed amongst seven subfamilies (TRPC, TRPV, TRPM, TRPML, TRPP, TRPA, TRPN). They subserve a wide range of functions throughout the body, most notably, though by no means exclusively, in sensory transduction and in vascular smooth muscle. However, their precise physiological roles and mechanism of activation and regulation are still only gradually being revealed. Most TRP channels are subject to multiple modes of regulation, but a common theme amongst the TRPC/V/M subfamilies is their regulation by lipid messengers. Genetic evidence supports an excitatory role of diacylglycerol (DAG) for the dTRP's, although curiously only DAG metabolites (PUFAs) have been found to activate the Drosophila channels. TRPC2,3,6 and 7 are widely accepted as DAG-activated channels, although TRPC3 can also be regulated via a store-operated mechanism. More recently PIP2 has been shown to be required for activity of TRPV5, TRPM4,5,7 and 8, whilst it may inhibit TRPV1 and the dTRPs. Although compelling evidence for a direct interaction of DAG with the TRPC channels is lacking, mutagenesis studies have identified putative PIP2-interacting domains in the C-termini of several TRPV and TRPM channels.

Animals↗

A ribonucleotide reductase homolog of cytomegalovirus and endothelial cell tropism.

Human cytomegalovirus infects vascular tissues and has been associated with atherogenesis and coronary restenosis. Although established laboratory strains of human cytomegalovirus have lost the ability to grow on vascular endothelial cells, laboratory strains of murine cytomegalovirus retain this ability. With the use of a forward-genetic procedure involving random transposon mutagenesis and rapid phenotypic screening, we identified a murine cytomegalovirus gene governing endothelial cell tropism. This gene, M45, shares sequence homology to ribonucleotide reductase genes. Endothelial cells infected with M45-mutant viruses rapidly undergo apoptosis, suggesting that a viral strategy to evade destruction by cellular apoptosis is indispensable for viral growth in endothelial cells.

3T3 Cells↗

Cysteinyl leukotrienes stimulate gut absorption of food allergens to promote anaphylaxis in mice.

Food-specific immunoglobulin E (IgE) triggers life-threatening anaphylaxis; however, for unclear reasons, some people with food-specific IgE are asymptomatic upon allergen consumption. We studied strains of mice with different sensitivities to anaphylaxis when orally challenged with allergen to identify possible causes. In resistant C57BL/6 mice, intestinal goblet cells transported less food allergen than did anaphylaxis-susceptible strains, even before allergic sensitization. In a forward genetic screen, resistance was correlated with dipeptidase 1 (Dpep1) variants. DPEP1 is expressed in intestinal epithelium and catabolizes leukotriene D4 (LTD4). Blocking DPEP1 with cilastatin, deleting Dpep1, or administering LTD4 orally enhanced allergen transport in resistant mice. Conversely, pretreatment of susceptible mice with a synthesis inhibitor, zileuton, abrogated allergen absorption and oral anaphylaxis, indicating that this could be an approach to treating food allergy.

Animals↗

Agrobacterium tumefaciens integrates transfer DNA into single chromosomal sites of dimorphic fungi and yields homokaryotic progeny from multinucleate yeast.

The dimorphic fungi Blastomyces dermatitidis and Histoplasma capsulatum cause systemic mycoses in humans and other animals. Forward genetic approaches to generating and screening mutants for biologically important phenotypes have been underutilized for these pathogens. The plant-transforming bacterium Agrobacterium tumefaciens was tested to determine whether it could transform these fungi and if the fate of transforming DNA was suited for use as an insertional mutagen. Yeast cells from both fungi and germinating conidia from B. dermatitidis were transformed via A. tumefaciens by using hygromycin resistance for selection. Transformation frequencies up to 1 per 100 yeast cells were obtained at high effector-to-target ratios of 3,000:1. B. dermatitidis and H. capsulatum ura5 lines were complemented with transfer DNA vectors expressing URA5 at efficiencies 5 to 10 times greater than those obtained using hygromycin selection. Southern blot analyses indicated that in 80% of transformants the transferred DNA was integrated into chromosomal DNA at single, unique sites in the genome. Progeny of B. dermatitidis transformants unexpectedly showed that a single round of colony growth under hygromycin selection or visible selection of transformants by lacZ expression generated homokaryotic progeny from multinucleate yeast. Theoretical analysis of random organelle sorting suggests that the majority of B. dermatitidis cells would be homokaryons after the ca. 20 generations necessary for colony formation. Taken together, the results demonstrate that A. tumefaciens efficiently transfers DNA into B. dermatitidis and H. capsulatum and has the properties necessary for use as an insertional mutagen in these fungi.

Agrobacterium tumefaciens↗

The small heart mutation reveals novel roles of Na+/K+-ATPase in maintaining ventricular cardiomyocyte morphology and viability in zebrafish.

Forward genetic screens in zebrafish have been used to identify mutations in genes with important roles in organogenesis. One of these mutants, small heart, develops a diminutive and severely malformed heart and multiple developmental defects of the brain, ears, eyes, and kidneys. Using a positional cloning approach, we identify that the mutant gene encodes the zebrafish Na+/K+-ATPase alpha1B1 protein. Disruption of Na+/K+-ATPase alpha1B1 function via morpholino "knockdown" or pharmacological inhibition with ouabain phenocopies the mutant phenotype, in a dose-dependent manner. Heterozygosity for the mutation sensitizes embryos to ouabain treatment. Our findings present novel genetic and morphological details on the function of the Na+/K+-ATPase alpha1B1 in early cardiac morphogenesis and the pathogenesis of the small heart malformation. We demonstrate that the reduced size of the mutant heart is caused by dysmorphic ventricular cardiomyocytes and an increase in ventricular cardiomyocyte apoptosis. This study provides a new insight that Na+/K+-ATPase alpha1B1 is required for maintaining ventricular cardiomyocyte morphology and viability.

Abnormalities, Drug-Induced↗

Finding new clock components: past and future.

The molecular mechanism of circadian clocks has been unraveled primarily by the use of phenotype-driven (forward) genetic analysis in a number of model systems. We are now in a position to consider what constitutes a clock component, whether we can establish criteria for clock components, and whether we have found most of the primary clock components. This perspective discusses clock genes and how genetics, molecular biology, and biochemistry have been used to find clock genes in the past and how they will be used in the future.

Animals↗

Age- and stage-specific regulation patterns in the hematopoietic stem cell hierarchy.

The molecular mechanisms that regulate self-renewal and differentiation of very primitive hematopoietic stem and progenitor cells in vivo are still poorly understood. Despite the clinical relevance, even less is known about the mechanisms that regulate these cells in old animals. In a forward genetic approach, using quantitative trait linkage analysis in the mouse BXD recombinant inbred set, this study identified loci that regulate the genetic variation in the size of primitive hematopoietic cell compartments of young and old C57BL6 and DBA/2 animals. Linked loci were confirmed through the generation and analysis of congenic animals. In addition, a comparative linkage analysis revealed that the number of primitive hematopoietic cells and hematopoietic stem cells are regulated in a stage-specific and an age-specific manner.

Aging↗

Novel memory mutants in Drosophila: behavioral characteristics of the mutant nemyP153.

BACKGROUND: Starting from Benzer's initiative, the approach of forward genetics has been widely used to isolate mutations affecting learning and memory. For this aim, mainly the odor-shock conditioning was employed. We have isolated P insertional mutations affecting memory after courtship conditioning - another form of classical conditioning in Drosophila. Here we report the behavioral characteristics of one of these mutants, which we have called nemy (no extended memory). RESULTS: The courtship activity of Drosophila males is reduced when a male has a previous experience of courting a fertilized female. In the wild-type strain C-S (K), this conditioned courtship inhibition lasts for 1-3 h in the test with a virgin female, and at least for 8 h in the test with a subsequent fertilized female. The mutant males nemyP153 display distinct memory deficiency in both tests already 0.5 h after training. The mutant males show an increased level of locomotor activity unrelated to courtship, and spend more time in such an element of courtship as pursuit. This, however, seems to be a pleiotropic effect of the mutation, independent from its influence on the courtship conditioning. The mutation reduces also memory performance after the odor-shock classical conditioning. At the same time, the sensory and motor functions involved in this type of learning seem to be normal. CONCLUSIONS: Insertion of P-lacW vector into 49B region of the second chromosome (mutation nemyP153) causes an increased level of locomotor activity, memory deficiency after the courtship conditioning and subnormal acquisition after the odor-shock conditioning.

Animals↗

Variation in the cortical area map of C57BL/6J and DBA/2J inbred mice predicts strain identity.

BACKGROUND: Recent discoveries suggest that arealization of the mammalian cortical sheet develops in a manner consonant with principles established for embryonic patterning of the body. Signaling centers release morphogens that determine regional growth and tissue identity by regulating regional expression of transcription factors. Research on mouse cortex has identified several candidate morphogens that affect anteroposterior or mediolateral cortical regionalization as well as mitogenesis. Inbred strains of laboratory mice can be exploited to study cortical area map formation if there are significant phenotypic differences with which to correlate gene polymorphism or expression data. Here we describe differences in the cortical area map of two commonly used inbred strains of laboratory mice, C57BL/6J and DBA/2J. Complete cortical hemispheres from adult mice were dissected and stained for the cytochrome oxidase enzyme in order to measure histochemically defined cortical areas. RESULTS: C57BL/6J has the larger neocortex, relatively larger primary visual cortex (V1), but relatively smaller posterior medial barrel subfield of the primary somatosensory cortex (PMBSF). The sample of C57BL/6J and DBA/2J mice can be discriminated with 90% accuracy on the basis of these three size dimensions. CONCLUSION: C57BL/6J and DBA/2J have markedly different cortical area maps, suggesting that inbred strains harbor enough phenotypic variation to encourage a forward genetic approach to understanding cortical development, complementing other approaches.

Animals↗

Increased synaptic microtubules and altered synapse development in Drosophila sec8 mutants.

BACKGROUND: Sec8 is highly expressed in mammalian nervous systems and has been proposed to play a role in several aspects of neural development and function, including neurite outgrowth, calcium-dependent neurotransmitter secretion, trafficking of ionotropic glutamate receptors and regulation of neuronal microtubule assembly. However, these models have never been tested in vivo. Nervous system development and function have not been described after mutation of sec8 in any organism. RESULTS: We identified lethal sec8 mutants in an unbiased forward genetic screen for mutations causing defects in development of glutamatergic Drosophila neuromuscular junctions (NMJs). The Drosophila NMJ is genetically malleable and accessible throughout development to electrophysiology and immunocytochemistry, making it ideal for examination of the sec8 mutant synaptic phenotype. We developed antibodies to Drosophila Sec8 and showed that Sec8 is abundant at the NMJ. In our sec8 null mutants, in which the sec8 gene is specifically deleted, Sec8 immunoreactivity at the NMJ is eliminated but immunoblots reveal substantial maternal contribution in the rest of the animal. Contrary to the hypothesis that Sec8 is required for neurite outgrowth or synaptic terminal growth, immunocytochemical examination revealed that sec8 mutant NMJs developed more branches and presynaptic terminals during larval development, compared to controls. Synaptic electrophysiology showed no evidence that Sec8 is required for basal neurotransmission, though glutamate receptor trafficking was mildly disrupted in sec8 mutants. The most dramatic NMJ phenotype in sec8 mutants was an increase in synaptic microtubule density, which was approximately doubled compared to controls. CONCLUSION: Sec8 is abundant in the Drosophila NMJ. Sec8 is required in vivo for regulation of synaptic microtubule formation, and (probably secondarily) regulation of synaptic growth and glutamate receptor trafficking. We did not find any evidence that Sec8 is required for basal neurotransmission.

Animals↗

Zebrafish penner/lethal giant larvae 2 functions in hemidesmosome formation, maintenance of cellular morphology and growth regulation in the developing basal epidermis.

Epithelial cells are equipped with junctional complexes that are involved in maintaining tissue architecture, providing mechanical integrity and suppressing tumour formation as well as invasiveness. A strict spatial segregation of these junctional complexes leads to the polarisation of epithelial cells. In vertebrate epithelia, basally localised hemidesmosomes mediate stable adhesion between epithelial cells and the underlying basement membrane. Although components of hemidesmosomes are relatively well known, the molecular machinery involved in governing the formation of these robust junctions, remains elusive. Here, we have identified the first component of this machinery using a forward genetic approach in zebrafish as we show that the function of penner (pen)/lethal giant larvae 2 (lgl2) is necessary for hemidesmosome formation and maintenance of the tissue integrity in the developing basal epidermis. Moreover, in pen/lgl2 mutant, basal epidermal cells hyper-proliferate and migrate to ectopic positions. Of the two vertebrate orthologues of the Drosophila tumour suppressor gene lethal giant larvae, the function of lgl2 in vertebrate development and organogenesis remained unclear so far. Here, we have unravelled an essential function of lgl2 during development of the epidermis in vertebrates.

Amino Acid Sequence↗