Mutagenesis by imprecise excision of the piggyBac transposon in Drosophila melanogaster

Mutagenesis by transposon-mediated imprecise excision is the most extensively used technique for mutagenesis in Drosophila. Although P-element is the most widely used transposon in Drosophila to generate deletion mutants, it is limited by the insertion coldspots in the genome where P-elements are rarely found. The piggyBac transposon was developed as an alternative mutagenic vector for mutagenesis of non-P-element targeted genes in Drosophila because the piggyBac transposon can more randomly integrate into the genome. Previous studies suggested that the piggyBac transposon always excises precisely from the insertion site without initiating a deletion or leaving behind an additional footprint. This unique characteristic of the piggyBac transposon facilitates reversible gene-transfer in several studies, such as the generation of induced pluripotent stem (iPS) cells from fibroblasts. However, it also raised a potential limitation of its utility in generating deletion mutants in Drosophila. In this study, we report multiple imprecise excisions of the piggyBac transposon at the sepiapterin reductase (SR) locus in Drosophila. Through imprecise excision of the piggyBac transposon inserted in the 5'-UTR of the SR gene, we generated a hypomorphic mutant allele of the SR gene which showed markedly decreased levels of SR expression. Our finding suggests that it is possible to generate deletion mutants by piggyBac transposon-mediated imprecise excision in Drosophila. However, it also suggests a limitation of piggyBac transposon-mediated reversible gene transfer for the generation of induced pluripotent stem (iPS) cells.     

利用果蝇模型研究人类心脏早期发育的分子机理(英文)

近年来 ,果蝇心脏特化的遗传机制已初步研究清楚 ,但控制人类心脏早期发育的基因尚待鉴定。因为调控果蝇和脊椎动物早期心脏细胞命运定型的途径具有保守性 ,果蝇是一种探讨人类心脏早期发育的分子机理的理想动物模式。为此目的 ,我们采用P转座子和EMS诱变技术建立了约 3 0 0 0个隐性致死基因平衡系。通过心脏前体细胞特异性抗体免疫组化筛选 ,我们检出 2 0 0余个表现心脏突变表型的平衡致死系。我们进一步利用RNAi技术对一些基因的功能进行了初步的研究 ,证明这些基因表现RNAi的突变表型 ,该类突变表型与基因突变时表现的表型相似 ,即心管呈缺陷型或无心脏前体细胞形成。利用果蝇和人类基因组计划获得的成果 ,我们从果蝇心脏侯选基因中初步克隆和鉴定了 5 0个人类同源基因 ,其中 2 0个是新基因。Northen印迹分析表明 ,一部分人类基因在心脏组织中有表达 ,从而为研究这些基因在人类心脏早期发育中的作用提供了信息。目前 ,我们正在建立转基因果蝇 ,以此为模型研究这些基因是否对心肌细胞发生或心肌功能起调控作用。产生心肌细胞突变类型的基因如果类似于人类心脏病综合症 ,则可以作为人类心脏疾病侯选基因作进一步的分析。     

Super-sized deletions: Improved transposon excision screens using a mus309 mutant background

Over the past two decades, a large collection of transposable elements inserted at various locations in the Drosophila melanogaster genome has been assembled. These transposons are frequently utilized in imprecise excision screens to generate deletions in genes of interest. In general, these screens involve genetic manipulations to combine a non-autonomous transposon and the appropriate transposase in individual male or female flies. DNA double-strand breaks are created via transposase action in both somatic and germline cells of these individuals and inaccurate repair events are recovered in the progeny. Because deletion-prone repair of transposon-induced double-strand breaks is rare, these screens generally require a significant investment of time and resources. We recently reported that conducting imprecise excision screens in mus309 mutant flies, which lack the Drosophila ortholog of the Bloom Syndrome helicase, results in an increase in both the number and size of deletions recovered. Here, we provide additional information for Drosophila researchers wishing to utilize this technique. In addition, we discuss how the general principle behind this technique can be applied in other contexts where double-strand breaks are being generated for the purpose of genome modification.     

The Drosophila slamdance gene: a mutation in an aminopeptidase can cause seizure,paralysis and neuronal failure

We report here the characterization of slamdance (sda), a Drosophila melanogaster "bang-sensitive" (BS) paralytic mutant. This mutant exhibits hyperactive behavior and paralysis following a mechanical "bang" or electrical shock. Electrophysiological analyses have shown that this mutant is much more prone to seizure episodes than normal flies because it has a drastically lowered seizure threshold. Through genetic mapping, molecular cloning, and RNA interference, we have demonstrated that the sda phenotype can be attributed to a mutation in the Drosophila homolog of the human aminopeptidase N (APN) gene. Furthermore, using mRNA in situ hybridization and LacZ staining, we have found that the sda gene is expressed specifically in the central nervous system at particular developmental stages. Together, these results suggest that the bang sensitivity in sda mutants is caused by a defective APN gene that somehow increases seizure susceptibility. Finally, by using the sda mutation as a sensitized background, we have been able to identify a rich variety of sda enhancers and other independent BS mutations.     

A novel genetic tool for clonal analysis of fourth chromosome mutations

The fourth chromosome of Drosophila remains one of the most intractable regions of the fly genome to genetic analysis. The main difficulty posed to the genetic analyses of mutations on this chromosome arises from the fact that it does not undergo meiotic recombination, which makes recombination mapping impossible, and also prevents clonal analysis of mutations, a technique which relies on recombination to introduce the prerequisite recessive markers and FLP-recombinase recognition targets (FRT). Here we introduce a method that overcomes these limitations and allows for the generation of single Minute haplo-4 clones of any fourth chromosome mutant gene in tissues of developing and adult flies.     

A novel genetic tool for clonal analysis of fourth chromosome mutations

The fourth chromosome of Drosophila remains one of the most intractable regions of the fly genome to genetic analysis. The main difficulty posed to the genetic analyses of mutations on this chromosome arises from the fact that it does not undergo meiotic recombination, which makes recombination mapping impossible, and also prevents clonal analysis of mutations, a technique which relies on recombination to introduce the prerequisite recessive markers and FLP-recombinase recognition targets (FRT). Here we introduce a method that overcomes these limitations and allows for the generation of single Minute haplo-4 clones of any fourth chromosome mutant gene in tissues of developing and adult flies.     

Analysis of a null mutation in the Drosophila splicing regulator Tra2 suggests its function is restricted to sexual differentiation

Tra2 is a regulator of pre-mRNA splicing and a key component of the Drosophila somatic sex determination pathway. Functional orthologs of this protein are thought to perform nonsex-specific functions essential for viability in both vertebrates and nematodes. Although Drosophila Tra2 is expressed throughout the soma of both sexes, studies on it have focused only on the sex-specific phenotypes of known viable alleles. Here we show that that widely used tra2 mutant alleles have residual activity and are not suitable for evaluating its effect on viability. To test whether Tra2 has an essential role in development, we generated a transposon-induced deletion in critical coding sequences. We find that tra2 deletion adults can survive as well as their heterozygous siblings. Thus, in contrast to other organisms, Tra2 is not required in Drosophila for general viability under laboratory conditions.     

Impact of the Chromatin Remodeling Factor CHD1 on Gut Microbiome Composition of Drosophila melanogaster

The composition of the intestinal microbiota of Drosophila has been studied in some detail in recent years. Environmental, developmental and host-specific genetic factors influence microbiome composition in the fly. Our previous work has indicated that intestinal bacterial load can be affected by chromatin-targeted regulatory mechanisms. Here we studied a potential role of the conserved chromatin assembly and remodeling factor CHD1 in the shaping of the gut microbiome in Drosophila melanogaster. Using high-throughput sequencing of 16S rRNA gene amplicons, we found that Chd1 deletion mutant flies exhibit significantly reduced microbial diversity compared to rescued control strains. Specifically, although Acetobacteraceae dominated the microbiota of both Chd1 wild-type and mutant guts, Chd1 mutants were virtually monoassociated with this bacterial family, whereas in control flies other bacterial taxa constituted ~20% of the microbiome. We further show age-linked differences in microbial load and microbiota composition between Chd1 mutant and control flies. Finally, diet supplementation experiments with Lactobacillus plantarum revealed that, in contrast to wild-type flies, Chd1 mutant flies were unable to maintain higher L. plantarum titres over time. Collectively, these data provide evidence that loss of the chromatin remodeler CHD1 has a major impact on the gut microbiome of Drosophila melanogaster.     

Genomic organization and characterization of the white locus of the Mediterranean fruitfly, Ceratitis capitata

An approximately 14-kb region of genomic DNA encoding the wild-type white eye (w+) color gene from the medfly, Ceratitis capitata has been cloned and characterized at the molecular level. Comparison of the intron-exon organization of this locus among several dipteran insects reveals distinct organizational patterns that are consistent with the phylogenetic relationships of these flies and the dendrogram of the predicted primary amino acid sequence of the white loci. An examination of w+ expression during medfly development has been carried out, displaying overall similarity to corresponding studies for white gene homologues in Drosophila melanogaster and other insects. Interestingly, we have detected two phenotypically neutral allelic forms of the locus that have arisen as the result of an apparently novel insertion or deletion event located in the large first intron of the medfly white locus. Cloning and sequencing of two mutant white alleles, w1 and w2, from the we,wp and M245 strains, respectively, indicate that the mutant conditions in these strains are the result of independent events--a frameshift mutation in exon 6 for w1 and a deletion including a large part of exon 2 in the case of w2.     

Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase

We have developed a novel technique, named DamID, for the identification of DNA loci that interact in vivo with specific nuclear proteins in eukaryotes. By tethering Escherichia coli DNA adenine methyltransferase (Dam) to a chromatin protein, Dam can be targeted in vivo to native binding sites of this protein, resulting in local DNA methylation. Sites of methylation can subsequently be mapped using methylation-specific restriction enzymes or antibodies. We demonstrate the successful application of DamID both in Drosophila cell cultures and in whole flies. When Dam is tethered to the DNA-binding domain of GAL4, targeted methylation is limited to a region of a few kilobases surrounding a GAL4 binding sequence. Using DamID, we identified a number of expected and unexpected target loci for Drosophila heterochromatin protein 1. DamID has potential for genome-wide mapping of in vivo targets of chromatin proteins in various eukaryotes.     

UGA nonsense mutation in the alcohol dehydrogenase gene of Drosophila melanogaster

A mutant gene, which we have designated AdhnB, codes for a defective form of the enzyme alcohol dehydrogenase in Drosophila melanogaster. We show that the polypeptide encoded by AdhnB is approximately 2000 Mr smaller than the protein synthesized under the direction of the wild-type alcohol dehydrogenase gene. In contrast, the alcohol dehydrogenase mRNA produced by both genes is the same size. We cloned and sequenced a portion of the protein-coding region of AdhnB and compared it to the same region in the wild-type gene. We found a single base substitution: a change of the TGG tryptophan codon at amino acid 235 to a TGA termination codon. This nonsense mutation accounts for the observed reduction in size of the alcohol dehydrogenase polypeptide. In further studies, we found that the steady-state levels of alcohol dehydrogenase mRNA in flies carrying the AdhnB gene and the wild-type alcohol dehydrogenase gene were indistinguishable. However, the steady-state level of alcohol dehydrogenase polypeptide was reduced to 1% of wild-type levels in flies with the AdhnB gene. Moreover, the rate of alcohol dehydrogenase synthesis in mutant flies was reduced to 50% of that found in wild type. The aberration in AdhnB thus affects both the rate of synthesis and the rate of degradation of the alcohol dehydrogenase peptide. AdhnB is the first reported nonsense mutant in Drosophila.     

Tuberous sclerosis complex and Myc coordinate the growth and division of Drosophila intestinal stem cells

Intestinal stem cells (ISCs) in the adult Drosophila melanogaster midgut can respond to damage and support repair. We demonstrate in this paper that the tuberous sclerosis complex (TSC) plays a critical role in balancing ISC growth and division. Previous studies have shown that imaginal disc cells that are mutant for TSC have increased rates of growth and division. However, we report in this paper that loss of TSC in the adult Drosophila midgut results in the formation of much larger ISCs that have halted cell division. These mutant ISCs expressed proper stem cell markers, did not differentiate, and had defects in multiple steps of the cell cycle. Slowing the growth by feeding rapamycin or reducing Myc was sufficient to rescue the division defect. The TSC mutant guts had a thinner epithelial structure than wild-type tissues, and the mutant flies were more susceptible to tissue damage. Therefore, we have uncovered a context-dependent phenotype of TSC mutants in adult ISCs, such that the excessive growth leads to inhibition of division.     

Microfluorometer assay to measure the expression of beta-galactosidase and green fluorescent protein reporter genes in single Drosophila flies

beta-galactosidase and green fluorescent protein (GFP) are among the most commonly used reporter genes to monitor gene expression in various organisms including Drosophila melanogaster. Their expression is usually detected in a qualitative way by direct microscopic observations of cells, tissues, or whole animals. To measure in vivo the inducibility of two antimicrobial peptide genes expressed during the Drosophila innate immune response, we have adapted two reporter gene systems based on the beta-galactosidase enzymatic activity and GFP. We have designed a 96-well microplate fluorometric assay sensitive enough to quantify the expression of both reporter genes in single flies. The assay has enabled us to process efficiently and rapidly a large number of individual mutant flies generated during an ethylmethane sulfonate saturation mutagenesis of the Drosophila genome. This method may be used in any screen that requires the quantification of reporter gene activity in individual insects.     

Gene deletions by ends-in targeting in Drosophila melanogaster

Following the advent of a gene targeting technique in Drosophila, different methods have been developed to modify the Drosophila genome. The initial demonstration of gene targeting in flies used an ends-in method, which generates a duplication of the target locus. The duplicated locus can then be efficiently reduced to a single copy by generating a double-strand break between the duplicated segments. This method has been used to knock out target genes by introducing point mutations. A derivative of this method is reported here. By using different homologous regions for the targeting and reduction steps, a complete deletion of the target gene can be generated to produce a definitive null allele. The breakpoints of the deletion can be precisely controlled. Unlike ends-out targeting, this method does not leave exogenous sequence at the deleted locus. Three endogenous genes, Sir2, Sirt2, and p53 have been successfully deleted using this method.     

The SR protein B52/SRp55 is essential for Drosophila development.

B52, also called SRp55, is a 52-kDa member of the Drosophila SR protein family of general splicing factors. Escherichia coli-produced B52 is capable of both activating splicing and affecting the alternative splice site choice in human in vitro splicing reactions. Here we report the isolation of a B52 null mutant generated by remobilizing a P element residing near the B52 gene. The resulting deletion, B52(28), is confined to the B52 gene and its neighbor the Hrb87F gene. Second-instar larvae homozygous for the deletion are deficient in both B52 mRNA and protein. The B52 null mutant is lethal at the first- and second-instar larval stages. Germ line transformation of Drosophila flies with B52 genomic DNA rescues this lethality. Thus, B52 is an essential gene and has a critical role in Drosophila development. Larvae deficient in B52 are still capable of splicing the five endogenous pre-mRNAs tested here, including both constitutively and alternatively spliced genes. Therefore, B52 is not required for all splicing in vivo. This is the first in vivo deficiency analysis of a member of the SR protein family.     

Quantitative real-time PCR for rapid and accurate titration of recombinant baculovirus particles

We describe the use of quantitative PCR (QPCR) to titer recombinant baculoviruses. Custom primers and probe were designed to gp64 and used to calculate a standard curve of QPCR derived titers from dilutions of a previously titrated baculovirus stock. Each dilution was titrated by both plaque assay and QPCR, producing a consistent and reproducible inverse relationship between C(T) and plaque forming units per milliliter. No significant difference was observed between titers produced by QPCR and plaque assay for 12 recombinant viruses, confirming the validity of this technique as a rapid and accurate method of baculovirus titration.