An efficient promoter trap for detection of patterned gene expression and subsequent functional analysis in Drosophila.
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ABSTRACT:
SUBMITTER: Larsen C
PROVIDER: S-EPMC1635159 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Many promoters have been used to drive expression of heterologous transgenes in insects. One major obstacle in the study of non-model insects is the dearth of useful promoters for analysis of gene function. Here, we investigated whether the promoter of the immediate-early gene, ie1, from the Bombyx mori nucleopolyhedrovirus (BmNPV) could be used to drive efficient transgene expression in a wide variety of insects. We used a piggyBac-based vector with a 3xP3-DsRed transformation marker to generate a reporter construct; this construct was used to determine the expression patterns driven by the BmNPV ie1 promoter; we performed a detailed investigation of the promoter in transgene expression pattern in Drosophila melanogaster and in B. mori. Drosophila and Bombyx belong to different insect orders (Diptera and Lepidoptera, respectively); however, and to our surprise, ie1 promoter-driven expression was evident in several tissues (e.g., prothoracic gland, midgut, and tracheole) in both insects. Furthermore, in both species, the ie1 promoter drove expression of the reporter gene from a relatively early embryonic stage, and strong ubiquitous ie1 promoter-driven expression continued throughout the larval, pupal, and adult stages by surface observation. Therefore, we suggest that the ie1 promoter can be used as an efficient expression driver in a diverse range of insect species.
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Project description:Large quantities of DNA sequence information about plant genes are rapidly accumulating in public databases, but to progress from DNA sequence to biological function a mutant allele for each of the genes ideally should be available. Here we describe a gene trap construct that allowed us to disrupt transcribed genes with a high efficiency in Arabidopsis thaliana. In the T-DNA vector used, the expression of a bacterial reporter gene coding for neomycin phosphotransferase II (nptII) depends on the in vivo generation of a translation fusion upon the T-DNA integration into the Arabidopsis genome. Analysis of 20 selected transgenic lines showed that 12 lines are T-DNA insertion mutants. The disrupted genes analyzed encoded ribosomal proteins (three lines), aspartate tRNA synthase, DNA ligase, basic-domain leucine zipper DNA binding protein, ATP-binding cassette transporter, and five proteins of unknown function. Four tagged genes were new for Arabidopsis. The results presented here suggest that gene trapping, using nptII as a reporter gene, can be as high as 80% and opens novel perspectives for systematic gene tagging in A. thaliana.
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Project description:In animals, each sequence-specific transcription factor typically binds to thousands of genomic regions in vivo. Our previous studies of 20 transcription factors show that most genomic regions bound at high levels in Drosophila blastoderm embryos are known or probable functional targets, but genomic regions occupied only at low levels have characteristics suggesting that most are not involved in the cis-regulation of transcription. Here we use transgenic reporter gene assays to directly test the transcriptional activity of 104 genomic regions bound at different levels by the 20 transcription factors. Fifteen genomic regions were selected based solely on the DNA occupancy level of the transcription factor Kruppel. Five of the six most highly bound regions drive blastoderm patterns of reporter transcription. In contrast, only one of the nine lowly bound regions drives transcription at this stage and four of them are not detectably active at any stage of embryogenesis. A larger set of 89 genomic regions chosen using criteria designed to identify functional cis-regulatory regions supports the same trend: genomic regions occupied at high levels by transcription factors in vivo drive patterned gene expression, whereas those occupied only at lower levels mostly do not. These results support studies that indicate that the high cellular concentrations of sequence-specific transcription factors drive extensive, low-occupancy, nonfunctional interactions within the accessible portions of the genome.
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Project description:The piggyBac transposon was modified to generate gene trap constructs, which were then incorporated into the genome of the Asian malaria vector, Anopheles stephensi and remobilized through genetic crosses using a piggyBac transposase expressing line. A total of 620 remobilization events were documented, and 73 were further characterized at the DNA level to identify patterns in insertion site preferences, remobilization frequencies, and remobilization patterns. Overall, the use of the tetameric AmCyan reporter as the fusion peptide displayed a preference for insertion into the 5'-end of transcripts. Notably 183 - 44882 bp upstream of the An. stephensi v1.0 ab initio gene models, which demonstrated that the promoter regions for the genes of An. stephensi are further upstream of the 5'-proximal regions of the genes in the ab inito models than may be otherwise predicted. RNA-Seq transcript coverage supported the insertion of the splice acceptor gene trap element into 5'-UTR introns for nearly half of all insertions identified. The use of a gene trap element that prefers insertion into the 5'-end of genes supports the use of this technology for the random generation of knock-out mutants, as well as the experimental confirmation of 5'-UTR introns in An. stephensi.
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Project description:Developmental processes are highly dependent on transcriptional regulation by RNA polymerase II. The RNA polymerase II core promoter is the ultimate target of a multitude of transcription factors that control transcription initiation. Core promoters consist of core promoter motifs, e.g. the initiator, TATA box, and the downstream core promoter element (DPE), which confer specific properties to the core promoter. Here, we explored the importance of core promoter functions in the dorsal-ventral developmental gene regulatory network. This network includes multiple genes that are activated by different nuclear concentrations of Dorsal, an NFκB homolog transcription factor, along the dorsal-ventral axis. We show that over two-thirds of Dorsal target genes contain DPE sequence motifs, which is significantly higher than the proportion of DPE-containing promoters in Drosophila genes. We demonstrate that multiple Dorsal target genes are evolutionarily conserved and functionally dependent on the DPE. Furthermore, we have analyzed the activation of key Dorsal target genes by Dorsal, as well as by another Rel family transcription factor, Relish, and the dependence of their activation on the DPE motif. Using hybrid enhancer-promoter constructs in Drosophila cells and embryo extracts, we have demonstrated that the core promoter composition is an important determinant of transcriptional activity of Dorsal target genes. Taken together, our results provide evidence for the importance of core promoter composition in the regulation of Dorsal target genes.
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Project description:Bacillus subtilis is an attractive host for production of recombinant proteins. Promoters and expression plasmid backbones have direct impacts on the efficiency of gene expression. To screen and isolate strong promoters, a promoter trap vector pShuttleF was developed in this study. Using the vector, approximately 1000 colonies containing likely promoters from Bacillus licheniformis genomic DNA were obtained. Amongst them, pShuttle-09 exhibited the highest ?-Gal activities in both Escherichia coli and B. subtilis. The activity of pShuttle-09 in B. subtilis was eight times of that of the P43 promoter, a commonly used strong promoter for B. subtilis. A sequence analysis showed that pShuttle-09 contained P(luxS) and truncated luxS in-frame fused with the reporter gene as well as another fragment upstream of P(luxS) containing a putative promoter. This putative promoter was a hybrid promoter and its ?-Gal activity was higher than P(luxS). Reconstructing the hybrid promoter from pShuttle-09 to P(lapS) further improved the ?-Gal production by 60%. The usefulness of our promoter trap system is likely due to random shuffling and recombination of DNA fragments and adoption of a rapid and high-throughput screening. Thus, our data provide additional evidence to support the concept of using a promoter trap system to create new promoters.
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Project description:Hemocytes, the immune cells in mosquitoes, participate in immune defenses against pathogens including malaria parasites. Mosquito hemocytes can also be infected by arthropod-borne viruses but the pro- or anti-viral nature of this interaction is unknown. Although there has been progress on hemocyte characterization during pathogen infection in mosquitoes, the specific contribution of hemocytes to immune responses and the hemocyte-specific functions of immune genes and pathways remain unresolved due to the lack of genetic tools to manipulate gene expression in these cells specifically. Here, we used the Gal4-UAS system to characterize the activity of the Drosophila hemocyte-specific hemolectin promoter in the adults of Anopheles gambiae, the malaria mosquito. We established an hml-Gal4 driver line that we further crossed to a fluorescent UAS responder line, and examined the expression pattern in the adult progeny driven by the hml promoter. We show that the hml regulatory region drives hemocyte-specific transgene expression in a subset of hemocytes, and that transgene expression is triggered after a blood meal. The hml promoter drives transgene expression in differentiating prohemocytes as well as in differentiated granulocytes. Analysis of different immune markers in hemocytes in which the hml promoter drives transgene expression revealed that this regulatory region could be used to study phagocytosis as well as melanization. Finally, the hml promoter drives transgene expression in hemocytes in which o'nyong-nyong virus replicates. Altogether, the Drosophila hml promoter constitutes a good tool to drive transgene expression in hemocyte only and to analyze the function of these cells and the genes they express during pathogen infection in Anopheles gambiae.
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Project description:External development and optical transparency of embryos make zebrafish exceptionally suitable for in vivo insertional mutagenesis using fluorescent proteins to visualize expression patterns of mutated genes. Recently developed Gene Breaking Transposon (GBT) vectors greatly improve the fidelity and mutagenicity of transposon-based gene trap vectors.We constructed and tested a bipartite GBT vector with Gal4-VP16 as the primary gene trap reporter. Our vector also contains a UAS:eGFP cassette for direct detection of gene trap events by fluorescence. To confirm gene trap events, we generated a UAS:mRFP tester line. We screened 270 potential founders and established 41 gene trap lines. Three of our gene trap alleles display homozygous lethal phenotypes ranging from embryonic to late larval: nsf(?tpl6), atp1a3a(tpl10) and flr(tpl19). Our gene trap cassette is flanked by direct loxP sites, which enabled us to successfully revert nsf(?tpl6), atp1a3a(tpl10) and flr(tpl19) gene trap alleles by injection of Cre mRNA. The UAS:eGFP cassette is flanked by direct FRT sites. It can be readily removed by injection of Flp mRNA for use of our gene trap alleles with other tissue-specific GFP-marked lines. The Gal4-VP16 component of our vector provides two important advantages over other GBT vectors. The first is increased sensitivity, which enabled us to detect previously unnoticed expression of nsf in the pancreas. The second advantage is that all our gene trap lines, including integrations into non-essential genes, can be used as highly specific Gal4 drivers for expression of other transgenes under the control of Gal4 UAS.The Gal4-containing bipartite Gene Breaking Transposon vector presented here retains high specificity for integrations into genes, high mutagenicity and revertibility by Cre. These features, together with utility as highly specific Gal4 drivers, make gene trap mutants presented here especially useful to the research community.
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Project description:Type 1 cannabinoid receptor blockers increase high-density lipoprotein cholesterol levels. Although genetic variation in the type 1 cannabinoid receptor--encoded by the CNR1 gene--is known to influence high-density lipoprotein cholesterol level as well, human studies conducted to date have been limited to genetic markers such as haplotype-tagging single nucleotide polymorphisms. Here we identify rs806371 in the CNR1 promoter as the causal variant. We re-sequence the CNR1 gene and genotype all variants in a DNA biobank linked to comprehensive electronic medical records. By testing each variant for association with high-density lipoprotein cholesterol level in a clinical practice-based setting, we localize a putative functional allele to a 100-bp window in the 5'-flanking region. Assessment of variants in this window for functional impact on electrophoretic mobility shift assay identifies rs806371 as a novel regulatory binding element. Reporter gene assays confirm that rs806371 reduces gene expression, thereby linking CNR1 gene variation to high-density lipoprotein cholesterol level in humans.
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Project description:BACKGROUND: Apolipoprotein E, a component of the plasma lipoproteins, plays an important role in the transport and metabolism of cholesterol and other lipids. Three single nucleotide polymorphisms (SNPs) -491A>T, -219T>G and +113G>C in the regulatory region of human apolipoprotein E gene (APOE) change the promoter activity and are associated with a wide variety of disorders including Alzheimer disease (AD). Functional SNPs in porcine APOE gene 5' regulatory region have not been explored. RESULTS: We examined SNPs within this region (from -831 to +855), and the analysis revealed that the T>A SNP at position -155 among these three polymorphism sites (-440, -155, +501) was found to exert a marked influence on the transcription of the porcine APOE gene. Electrophoretic mobility shift assays showed that the binding affinity of oligonucletides containing the -155A to transcription factor(s) was stronger than that of the -155T. Transient transfection assays and quantitative real-time PCR results revealed that the -155T>A variant enhanced the activity of the APOE promoter and was associated with increased APOE mRNA levels in vivo. CONCLUSIONS: These data suggest that the -155T>A mutation in the promoter region of the porcine APOE gene is an important functional variant. The results provided new insights into aspects of pig genetics and might also facilitate the application of pigs in biomedical studies addressing important human diseases.
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