Genetic engineering technologies can be used both to create transgenic mosquitoes carrying antipathogen effector genes targeting human malaria parasites and to generate gene-drive systems capable of introgressing the genes throughout wild vector populations. We developed a highly effective autonomous Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)-mediated gene-drive system in the Asian malaria vector Anopheles stephensi, adapted from the mutagenic chain reaction (MCR). This specific system results in progeny of males and females derived from transgenic males exhibiting a high frequency of germ-line gene conversion consistent with homology-directed repair (HDR). This system copies an ∼17-kb construct from its site of insertion to its homologous chromosome in a faithful, site-specific manner. Dual anti-Plasmodium falciparum effector genes, a marker gene, and the autonomous gene-drive components are introgressed into ∼99.5% of the progeny following outcrosses of transgenic lines to wild-type mosquitoes. The effector genes remain transcriptionally inducible upon blood feeding. In contrast to the efficient conversion in individuals expressing Cas9 only in the germ line, males and females derived from transgenic females, which are expected to have drive component molecules in the egg, produce progeny with a high frequency of mutations in the targeted genome sequence, resulting in near-Mendelian inheritance ratios of the transgene. Such mutant alleles result presumably from nonhomologous end-joining (NHEJ) events before the segregation of somatic and germ-line lineages early in development. These data support the design of this system to be active strictly within the germ line. Strains based on this technology could sustain control and elimination as part of the malaria eradication agenda.
Sestrins are conserved proteins that accumulate in cells exposed to stress and potentiate adenosine monophosphate-activated protein kinase (AMPK) and inhibit activation of target of rapamycin (TOR). We show that abundance of Drosophila Sestrin (dSesn) is increased upon chronic TOR activation through accumulation of reactive oxygen species (ROS) that cause activation of c-Jun N-terminal kinase (JNK) and transcription factor FoxO (Forkhead box O). Loss of dSesn resulted in age-associated pathologies including triglyceride accumulation, mitochondrial dysfunction, muscle degeneration and cardiac malfunction, which were prevented by pharmacological activation of AMPK or inhibition of TOR. Hence, dSesn appears to be a negative feedback regulator of TOR that integrates metabolic and stress inputs and prevents pathologies caused by chronic TOR activation, that may result from diminished autophagic clearance of damaged mitochondria, protein aggregates, or lipids.TOR (target of rapamycin) is a key protein kinase that regulates cell growth and metabolism to maintain cellular and organismal homeostasis (1-3). Insulin (Ins) and insulin-like growth factors (IGF) are major TOR activators that operate through phosphoinositide 3-kinase (PI3K) and the protein kinase AKT (2). Conversely, adenosine monophosphate activated protein kinase (AMPK), which is activated upon energy depletion, caloric restriction (CR), or genotoxic damage, is a stress-responsive inhibitor of TOR activation (2,4). TOR stimulates cell growth and anabolism by increasing protein and lipid synthesis through p70 S6 kinase (S6K), eukaryotic translation initiation factor 4E-binding protein (4E-BP), and sterol response element binding protein (SREBP) (1-3,5) and by decreasing autophagic † To whom correspondence should be addressed. karinoffice@ucsd.edu. Prolonged TOR signaling induces dSesnPersistent TOR activation in wing discs by a constitutively active form of insulin receptor (InR CA ) resulted in prominent dSesn protein accumulation, not seen in a dSesn-null larvae (Fig. 1, A to C). InR CA also induced accumulation of dSesn RNA (Fig. 1, D to F), indicating that dSesn accumulation is due to increased transcription or mRNA stabilization. As dSesn accumulation was restricted to cells in which TOR was activated, the response is likely to be cell autonomous. dSesn was also induced when TOR was chronically activated by overexpression of the small guanine triphosphatase Rheb (Fig. 1G), or clonal loss of PTEN (phosphatase and tensin homolog) or TSC1 (tuberous sclerosis complex 1) (Fig. 1, H TOR signaling generates ROS to induce dSesnIn mammals, transcription of Sesn genes is increased in cells exposed to oxidative stress (9,11) and we observed ROS accumulation, detected by oxidation of dihydroethidium (DHE), in the same region of the imaginal discs in which InR CA or Rheb were expressed (Fig. 2, A (Fig. 2F).FoxO and p53 are ROS-activated transcription factors that control mammalian Sesn genes (9-12,14). The dSesn locus contains 8 perfect FoxO-response elemen...
An organism with a single recessive loss-of-function allele will typically have a wild-type phenotype while individuals homozygous for two copies of the allele will display a mutant phenotype. Here, we develop a method that we refer to as the mutagenic chain reaction (MCR), which is based on the CRISPR/Cas9 genome editing system for generating autocatalytic mutations to generate homozygous loss-of-function mutations. We demonstrate in Drosophila that MCR mutations efficiently spread from their chromosome of origin to the homologous chromosome thereby converting heterozygous mutations to homozygosity in the vast majority of somatic and germline cells. MCR technology should have broad applications in diverse organisms.
We performed a systematic BLAST analysis of 929 human disease gene entries associated with at least one mutant allele in the Online Mendelian Inheritance in Man (OMIM) database against the recently completed genome sequence of Drosophila melanogaster. The results of this search have been formatted as an updateable and searchable on-line database called Homophila. Our analysis identified 714 distinct human disease genes (77% of disease genes searched) matching 548 unique Drosophila sequences, which we have summarized by disease category. This breakdown into disease classes creates a picture of disease genes that are amenable to study usingDrosophila as the model organism. Of the 548Drosophila genes related to human disease genes, 153 are associated with known mutant alleles and 56 more are tagged byP-element insertions in or near the gene. Examples of how to use the database to identify Drosophila genes related to human disease genes are presented. We anticipate that cross-genomic analysis of human disease genes using the power of Drosophilasecond-site modifier screens will promote interaction between human andDrosophila research groups, accelerating the understanding of the pathogenesis of human genetic disease. The Homophila database is available at http://homophila.sdsc.edu.
Drosophila melanogaster is emerging as one of the most effective tools for analyzing the function of human disease genes, including those responsible for developmental and neurological disorders, cancer, cardiovascular disease, metabolic and storage diseases, and genes required for the function of the visual, auditory and immune systems. Flies have several experimental advantages, including their rapid life cycle and the large numbers of individuals that can be generated, which make them ideal for sophisticated genetic screens, and in future should aid the analysis of complex multigenic disorders. The general principles by which D. melanogaster can be used to understand human disease, together with several specific examples, are considered in this review.
A P-element vector has been constructed and used to generate lines of flies with single autosomal P-element insertions. The lines were analyzed in two ways: (1) the identification of cis-acting patterning information within the Drosophila genome, as revealed by a lacZ reporter gene within the P element, and (2) the isolation of lethal mutations. We examined 3768 independent lines for the expression of lacZ in embryos and looked among these lines for lethal mutations affecting embryonic neurogenesis. This type of screen appears to be an effective way to find new loci that may play a role in the development of the Drosophila nervous system.[Key Words: P element; lacZ; mutagenesis; cell market; Drosophila; pattern]Received May 30, 1989; revised version accepted July 11, 1989. One approach to studying development is to obtain genetic variants that are defective in some crucial step. This type of genetic analysis has been very successful in identifying virtually all of the zygotic loci required for the early stages of segmentation during embryogenesis in Drosophila melanogaster Niisslein-Volhard et al. 1984;Wieschaus et al. 1984). Besides chemical mutagenesis, transposon tagging has been used as a mutagen and allows rapid cloning of genes of interest (Bingham et al. 1981;Kidwell 1986).Recently, a scheme wherein single P elements are mobilized to new chromosomal locations has been implemented successfully (Cooley et al. 1988). The essential nature of this approach is to use two separate P elements to provide the two functions necessary for transposition. The first is a genetically marked P element that is defective in production of transposase but contains the ends required for its own transposition. The second is a P element with functional transposase activity but a much reduced likelihood for its own transposition (Robertson et al. 1988). Transposition of the marked P element then is initiated by crossing flies that carry only the marked P element to those that harbor only transposase. Insertions generated by this scheme are recovered in flies lacking tranposase activity and are therefore genetically stable.P-element vectors also have been used recently to search for cis-acting sequences which confer tissue-specific expression of a p-galactosidase [lacZ] fusion gene driven by the weak promoter of the P-element transpoPresent addresses:
Summary Chronic activation of mammalian target of rapamycin complex 1 (mTORC1) and p70 S6 kinase (S6K) in response to hypernutrition contributes to obesity-associated metabolic pathologies including hepatosteatosis and insulin resistance. Sestrins are stress-inducible proteins that activate AMP-activated protein kinase (AMPK) and suppress mTORC1-S6K activity, but their role in mammalian physiology and metabolism has not been investigated. We show that Sestrin2, encoded by the Sesn2 locus whose expression is induced upon hypernutrition, maintains metabolic homeostasis in liver of obese mice. Sesn2 ablation exacerbates obesity-induced mTORC1-S6K activation, glucose intolerance, insulin resistance and hepatosteatosis, all of which are reversed by AMPK activation. Furthermore, concomitant ablation of Sesn2 and Sesn3 provokes hepatic mTORC1-S6K activation and insulin resistance even in the absence of nutritional overload and obesity. These results demonstrate an important homeostatic function for the stress-inducible Sestrin protein family in the control of mammalian lipid and glucose metabolism.
rhomboid (rho) encodes a putative transmembrane receptor that is required for the differentiation of the ventral epidermis. It is initially expressed before the completion of cellularization in lateral stripes within the presumptive neuroectoderm. Here, we present evidence that the maternal morphogen dorsal (dl) acts in concert with basic helix-loop-helix (b-HLH) proteins, possibly including twist (twi), to activate rho in both lateral and ventral regions. Expression is blocked in ventral regions (the presumptive mesoderm) by sna/l (sna), which is also a direct target of the d/morphogen. A 300-bp region of the rho promoter (the NEE), which is sufficient for neuroectoderm expression, contains a cluster of d/and b-HLH activator sites that are closely linked to sna repressor sites. Mutations in these binding sites cause genetically predicted changes in the levels and limits of rho expression. In particular, the disruption of sna-binding sites causes a derepression of the pattern throughout ventral regions, providing evidence that sna is directly responsible for establishing the mesoderm/neuroectoderm boundary before gastrulation. The tight linkage of activator and repressor sites in the rho NEE is similar to the arrangement of binding sites observed in the even-skipped stripe 2 element, which is regulated by bicoid (bcal. This suggests that the d/and bcd morphogens use a similar mechanism to make stripes in the Drosophila embryo.
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