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Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T19:04:53Z No. of bitstreams: 1 S1415-47572004000300011.pdf: 311036 bytes, checksum: 449f90200aee786d72ccea388e2c8567 (MD5) Made available in DSpace on 2013-08-22T19:04:53Z (GMT). No. of bitstreams: 1 S1415-47572004000300011.pdf: 311036 bytes, checksum: 449f90200aee786d72ccea388e2c8567 (MD5) Previous issue date: 2004-01-01 Made available in DSpace on 2013-09-30T20:08:20Z (GMT). No. of bitstreams: 2 S1415-47572004000300011.pdf: 311036 bytes, checksum: 449f90200aee786d72ccea388e2c8567 (MD5) S1415-47572004000300011.pdf.txt: 21124 bytes, checksum: a05d0ac1d5b26411013c227dbc47d891 (MD5) Previous issue date: 2004-01-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:17:27Z No. of bitstreams: 2 S1415-47572004000300011.pdf: 311036 bytes, checksum: 449f90200aee786d72ccea388e2c8567 (MD5) S1415-47572004000300011.pdf.txt: 21124 bytes, checksum: a05d0ac1d5b26411013c227dbc47d891 (MD5) Made available in DSpace on 2014-05-20T15:17:27Z (GMT). No. of bitstreams: 2 S1415-47572004000300011.pdf: 311036 bytes, checksum: 449f90200aee786d72ccea388e2c8567 (MD5) S1415-47572004000300011.pdf.txt: 21124 bytes, checksum: a05d0ac1d5b26411013c227dbc47d891 (MD5) Previous issue date: 2004-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Previously, only partial P element sequences have been reported in the saltans group of Drosophila but in this paper we report two complete P element sequences from Drosophila sturtevanti and Drosophila prosaltans. The divergence of these sequences from the canonical P element of Drosophila melanogaster is about 31% at the nucleotide level. Phylogenetic analysis revealed that both elements belong to a clade of divergent sequences from the saltans and willistoni groups previously described by other authors. Universidade Estadual Paulista Departamento de Biologia Universidade Estadual Paulista Departamento de Biologia

P elements inserted near the left telomere of the X chromosome are associated with the P cytotype, a maternally transmitted condition that strongly regulates the activity of the P transposon family in some strains of Drosophila. The regulatory abilities of two such elements, TP5 and TP6, are stable in homozygous stocks over many generations. However, these regulatory abilities are attenuated when the telomeric P elements are transmitted through heterozygous females, and they are utterly lost when the elements are transmitted through males. Paternally transmitted telomeric P elements reacquire regulatory ability when they pass through a female germ line. This reacquisition is enhanced if the females in which it occurs came from mothers who carried a telomeric P element. The enhancement has two components: (1) a strictly maternal effect that is transmitted to the females independently of the mother's telomeric P element (“presetting” or the “pre-P cytotype”) and (2) a zygotic effect associated with inheritance of the mother's telomeric P element. One telomeric P element can enhance the reacquisition of another's regulatory ability. When X chromosomes that carry telomeric P elements are extracted through males and made homozygous by using a balancer chromosome, most of the resulting stocks develop strong regulatory abilities in a few generations. However, some of the stocks do not attain the regulatory ability of the original population.

The α-glycerophosphate (GP) cycle in dipteran insect flight muscle is composed of two enzyme, the cytoplasmic α-glycerophosphate dehydregenase (α GPDH) and the mitochondrial α-glycerophosphate oxidase (α GPO). In order to analyze the enzyme activity in transformant, nine strains of Drosophila melanogaster have been constructed to have the flight muscle-specific α -glycerophosphate dehydrogenase (α GPDH) cDNA under the control of the Drosophila heat shock promoter (hsp70) on the third chromosome. These strains were compared with α GPDH null mutant line in the enzyme activity. All the transgenic strains exhibited higher enzymatic activities than null mutant line, but the amount of activity was still very low even at heat shock condition showing 30% in α GPDH enzyme amount, compared with wild type.

Mushroom bodies (MB) are substructures in the Drosophila brain that are essential for memory. At present, MB anatomy is rather well described when compared to other brain areas, and elucidation of the genetic control of the development and projection patterns of MB neurons will be important to the understanding of their functions. We have performed a gain-of-function screen in order to identify genes that are involved in MB development. We drove expression of genes in MB neurons by crossing 2407 GAL4-driven UY element lines to lines containing an MB GAL4 source and UAS-GFP elements, and looked for defects in the MB structure. We have molecularly identified the genomic regions adjacent to the 26 positive UY insertions and found 18 potential genes that exhibit adult MB gain-of-function phenotypes. The proteins encoded by these candidate genes include, as well as genes with yet unknown function, transcription factors (e.g., tramtrack), nanos RNA-binding protein, microtubule-severing protein, vesicle trafficking proteins, axon guidance receptor, and the Src64 cytoplasmic protein tyrosine kinase. These genes are involved in key features of neuron cell biology. In three cases, tramtrack, nanos, and Src64, we show that the open reading frame located directly downstream of the UY P element is indeed the expressed target gene. Loss-of-function mutations of both ttk and Src64 lead to MB phenotypes proving that these genes are involved in the genetic control of MB development. Moreover, Src64 is shown here to act in a cell-autonomous fashion and is likely to interact with the previously-identified linotte/derailed receptor tyrosine kinase in MB development.

The P transposable element copy numbers and the KP/full-sized P element ratios were determined in eight Brazilian strains of Drosophila melanogaster. Strains from tropical regions showed lower overall P element copy numbers than did strains from temperate regions. Variable numbers of full-sized and defective elements were detected, but the full-sized P and KP elements were the predominant classes of elements in all strains. The full-sized P and KP element ratios were calculated and compared with latitude. The northernmost and southernmost Brazilian strains showed fewer full-sized elements than KP elements per genome, and the strains from less extreme latitudes had many more full-sized P than KP elements. However, no clinal variation was observed. Strains from different localities, previously classified as having P cytotype, displayed a higher or a lower proportion of KP elements than of full-sized P elements, as well as an equal number of the two element types, showing that the same phenotype may be produced by different underlying genomic components of the P-M system.

The isolation of chemically induced mutations in forward genetic screens is one of the hallmarks of Drosophila genetics. However, mapping the corresponding loci and identifying the molecular lesions associated with these mutations are often difficult and labor-intensive. Two mapping methods are most often used in flies: meiotic recombination mapping with marked chromosomes and deficiency mapping. The availability of the fly genome sequence allows the establishment and usage of molecular markers. Single-nucleotide polymorphisms have therefore recently been used to map several genes. Here we show that thousands of molecularly mapped P element insertions in fly strains that are publicly available provide a powerful alternative method to single-nucleotide polymorphism mapping. We present a strategy that allows mapping of lethal mutations, as well as viable mutations with visible phenotypes, with minimal resources. The most important unknown in using recombination rates to map at high resolution is how accurately recombination data correlate with molecular maps in small intervals. We therefore surveyed distortions of recombination rates in intervals P elements in Drosophila.

Insertion of a P element in one copy of the Drosophila Indy gene results in a significant increase in Drosophila lifespan. In order to examine whether mutations or polymorphisms in the human Indy gene promoted human longevity, the Indy gene was sequenced in 13 subjects of age 90-99, 12 subjects between age 38-49 and 18 subjectsage 23. Three coding region polymorphisms resulting in an amino acid change, F62L, V80L and I550V, two silent coding region polymorphisms, A99A and I551I, and three intron polymorphisms, IVS2+10 C deletion, IVS9+40T/A and IVS10+38C/T were identified. The I550V polymorphism occurred in whites, Hispanics, Asians and African-Americans at allele frequencies of 0.3514, 0.5104, 0.4274 and 0.5375, respectively. The I550V polymorphism was genotyped in 2366 subjects and examined for its association with age and weight.

Background: The identification of the function of all genes that contribute to specific biological processes and complex traits is one of the major challenges in the postgenomic era. One approach is to employ forward genetic screens in genetically tractable model organisms. In Drosophila melanogaster, P element-mediated insertional mutagenesis is a versatile tool for the dissection of molecular pathways, and there is an ongoing effort to tag every gene with a P element insertion. However, the vast majority of P element insertion lines are viable and fertile as homozygotes and do not exhibit obvious phenotypic defects, perhaps because of the tendency for P elements to insert 5′ of transcription units. Quantitative genetic analysis of subtle effects of P element mutations that have been induced in an isogenic background may be a highly efficient method for functional genome annotation.Results: Here, we have tested the efficacy of this strategy by assessing the extent to which screening for quantitative effects of P elements on sensory bristle number can identify genes affecting neural development. We find that such quantitative screens uncover an unusually large number of genes that are known to function in neural development, as well as genes with yet uncharacterized effects on neural development, and novel loci.Conclusions: Our findings establish the use of quantitative trait analysis for functional genome annotation through forward genetics. Similar analyses of quantitative effects of P element insertions will facilitate our understanding of the genes affecting many other complex traits in Drosophila.

The Sleeping Beauty (SB) transposon system, derived from teleost fish sequences, is extremely effective at delivering DNA to vertebrate genomes, including those of humans. We have examined several parameters of the SB system to improve it as a potential, nonviral vector for gene therapy. Our investigation centered on three features: the carrying capacity of the transposon for efficient integration into chromosomes of HeLa cells, the effects of overexpression of the SB transposase gene on transposition rates, and improvements in the activity of SB transposase to increase insertion rates of transgenes into cellular chromosomes. We found that SB transposons of about 6 kb retained 50% of the maximal efficiency of transposition, which is sufficient to deliver 70-80% of identified human cDNAs with appropriate transcriptional regulatory sequences. Overexpression inhibition studies revealed that there are optimal ratios of SB transposase to transposon for maximal rates of transposition, suggesting that conditions of delivery of the two-part transposon system are important for the best gene-transfer efficiencies. We further refined the SB transposase to incorporate several amino acid substitutions, the result of which led to an improved transposase called SB11. With SB11 we are able to achieve transposition rates that are about 100-fold above those achieved with plasmids that insert into chromosomes by random recombination. With the recently described improvements to the transposon itself, the SB system appears to be a potential gene-transfer tool for human gene therapy.

We report the identification of genomic sequences in various anopheline mosquitoes (family Culicidae: suborder Nematocera: order Diptera) showing homology to the class II, short inverted-terminal-repeat (ITR) transposable element P from Drosophila melanogaster (family Drosophilidae; suborder Brachycera: order Diptera). Anopheles gambiae appears to have at least six distinct P elements. Other anopheline species, including four additional members of the An. gambiae species complex (An. arabiensis, An. merus, An. melas and An. quadriannulatus), Anopheles stephensi (all subgenus Cellia), An. quadrimaculatus (subgenus Anopheles) and Anopheles albimanus (subgenus Nyssorhynchus) also possess P elements similar to those found in An. gambiae. Ten distinct P element types were identified in the genus Anopheles. At least two of the An. gambiae elements appears to be intact and potentially functional. Phylogenetic analysis of the anopheline P elements reveals them to belong to a distinctly different clade from the brachyceran P elements.

Tn5 is an excellent model system for understanding the molecular basis of DNA-mediated transposition. Mechanistic information has come from genetic and biochemical investigations of the transposase and its interactions with the recognition DNA sequences at the ends of the transposon. More recently, molecular structure analyses of catalytically active transposase; transposon DNA complexes have provided us with unprecedented insights into this transposition system. Transposase initiates transposition by forming a dimeric transposase, transposon DNA complex. In the context of this complex, the transposase then catalyses four phosphoryl transfer reactions (DNA nicking, DNA hairpin formation, hairpin resolution and strand transfer into target DNA) resulting in the integration of the transposon into its new DNA site. The studies that elucidated these steps also provided important insights into the integration of retroviral genomes into host DNA and the immune system V(D)J joining process. This review will describe the structures and steps involved in Tn5 transposition and point out a biologically important although surprising characteristic of the wild-type Tn5 transposase. Transposase is a very inactive protein. An inactive transposase protein ensures the survival of the host and thus the survival of Tn5.

The P-element is one of the best understood eukaryotic transposable elements. It invadedDrosophila melanogasterpopulations within a few decades, but was thought to be absent from close relatives, includingD. simulans. Five decades after the spread inD. melanogaster, we provide evidence that the P-element has also invadedD. simulans. P-elements inD. simulansappear to have been acquired recently fromD. melanogasterprobably via a single horizontal transfer event. Expression data indicate that the P-element is processed in the germline ofD. simulans, and genomic data show an enrichment of P-element insertions in putative origins of replication, similar to that seen inD. melanogaster. This ongoing spread of the P-element in natural populations provides an unique opportunity to understand the dynamics of transposable element spreads and the associated piRNA defense mechanisms.

The need for genetically engineered animals for drug target discovery, development and validation is growing. Transgenic animals, including mice and rats, and knockout and knock-in animals, provide useful animal models on which to test hypotheses and therapies. Tosk, Inc. has developed two gene delivery vectors based on the Drosophila P element transposon: StealthGene™ and a targeted gene delivery (TGD) vector that works via homologous recombination. The promise of these new vectors will be assessed for applications ranging from replacing current transgenic and knockout technologies, to creating large transgenic animals such as goats, pigs, sheep and chickens, to human gene therapy.

To identify genes expressed in interesting spatial and temporal patterns during development, we mobilized transposons carrying the yeast transcriptional activator, GAL4, to create new insertions throughout the Drosophila genome. At these new sites, neighboring enhancers drive expression of GAL4 in a pattern similar to that of nearby genes (Brand and Perrimon, 1993). Since GAL4 binds to a UAS target sequence, flies containing a GAL4 transposon can activate transcription of a UAS-linked gene, thereby resulting in controlled expression of that secondary gene. Thus, the GAL4 P element is a useful tool both for identifying genes of interest and for ectopically expressing genes in novel tissues or at specific developmental stages. We mobilized a GAL4 insertion on the second chromosome (P{GawB} CY2; Queenan et al., 1997) to generate a collection of GAL4-expressing enhancer trap lines. F1 “jumpstarter” males (w/Y; GAL4/CyO; Sb 2-3/ ) were mated in vials to attached-X (C(1)DX/Y) females. From 200 such crosses, red-eyed, curly-winged, long-bristled sons (bearing a new GAL4 insertion) were selected and mated individually to w/w females to establish 50 lines. Chromosome-mapping data, enhancer trap pattern, and other phenotypic characters demonstrated that four insertions were duplicates, yielding a total of 46 independent lines (Table 1). All X and third chromosome lines are viable, although three lines exhibit reduced viability or fertility (lines 15, 45, and 50). We could not determine the viability of the CyOlinked lines since the balancer chromosome is homozygous lethal. The genotypes of the resulting stocks are: X-chromosome lines, w P{GawB}, second-chromosome lines, y w; Pin/CyO, P{GawB}, and third chromosome lines, y w; III P{GawB}. Line 15 also carries the FM6, y w Bar balancer while lines 45 and 50 contain TM3, Sb Ser. Finally, line number three exhibits a striking eye phenotype consisting of a variable loss of eye pigment in a gradient along the anterior/posterior axis. Surprisingly, this line lacks detectable GAL4 expression in larval eye discs. We characterized sequences flanking a subset of the insertions in our collection by the inverse PCR method (http:www.fruitfly.org/methods/). Table 1 lists neighboring predicted or known genes. To identify and visualize the morphology of cells expressing GAL4, we crossed females from each line to males carrying UAS-taulacZ, which links the -galactosidase enzyme to the tubulin-binding protein Tau (Hildago et al., 1995). Some lines (1, 7, 10 (males only), 14, 22, 25, 26, 32, 35, 36, and 41) were lethal in pupal stages when crossed to this reporter; therefore, to assay ovary and testis expression, we crossed these strains to UAS-lacZ.NZ, which contains a nuclear-localization signal (Y. Hiromi and S. West, unpubl. results). Tables 1 and 2 describe the expression patterns for each line and Figure 1 shows selected patterns that illustrate key points. More detailed descriptions of all the patterns are available upon request. All lines exhibit interesting staining in at least one developmental stage. As commonly noted, we observe patchy or variable GAL4 expression in many lines. For example, GAL4 drives embryonic PNS expression in 18 lines (Table 1). While all embryos within a given strain exhibit a general PNS pattern that is consistent, within an individual embryo, slight variations often occur between segmental repeats (e.g., brackets in Fig. 1a). Only a few lines express GAL4 in a single tissue at a specific developmental stage. For example, line 20 drives expression in the longitudinal visceral mesoderm from stage 12 onwards (Fig. 1c). When crossed to UASGFP, line 20 can be used to visualize this highly migratory tissue in vivo. As Tables 1 and 2 indicate, many lines express GAL4 in identical tissues. Nevertheless, important differences may exist in the distribution of expression within each tissue. For example, Fig. 1f–i shows representative wing discs from four lines that express GAL4 in all imaginal discs, the brain, and the central nervous system (IGD, brain, and CNS). The distinct patterns shown in the wing discs presumably reveal specific regulatory elements that govern expression of nearby

A highly efficient method of transposon mutagenesis was developed for genetic analysis of Xanthobacter autotrophicus Py2. The method makes use of a transposon delivery vector that encodes a hyperactive Tn 5 transposase that is 1,000-fold more active than the wild-type transposase. In this construct, the transposase is expressed from the promoter of the tetA gene of plasmid RP4, which is functional in a wide variety of organisms. The transposon itself contains a kanamycin resistance gene as a selectable marker and the origin of replication from plasmid R6K to facilitate subsequent cloning of the resulting insertion site. To test the effectiveness of this method, mutants unable to produce the characteristic yellow pigment (zeaxanthin dirhamnoside) of X. autotrophicus Py2 were isolated and analyzed. Transposon insertions were obtained at high frequency: approximately 1 x 10(-3) per recipient cell. Among these, pigment mutants were observed at a frequency of approximately 10(-3). Such mutants were found to have transposon insertions in genes homologous to known carotenoid biosynthetic genes previously characterized in other pigmented bacteria. Mutants were also isolated in Pseudomonas stutzeri and in an Alcaligenes faecalis, demonstrating the effectiveness of the method in diverse Proteobacteria. Preliminary results from other laboratories have confirmed the effectiveness of this method in additional phylogenetically diverse species.

The bric a brac (bab) locus is required for proper development and morphogenesis of ovaries (Couderc et al., 2002; Godt and Laski, 1995; Sahut-Barnola, 1995), legs (Couderc et al., 2002; Chu et al., 2002; Godt and Laski, 1995), antennae (Chu et al., 2002; Godt et al., 1993) and abdomen (Couderc et al., 2002; Kopp et al., 2000) of Drosophila melanogaster. bab is a large locus covering 140 kb and is composed of two genes, bab1 and bab2, that are similar in sequence and structure and likely arose by a chromosomal duplication (Couderc et al., 2002). Both genes encode proteins with a BTB domain and a pipsqueak motif (Couderc et al., 2002), domains found in a number of transcriptional regulators (Collins, 2001; Lehmann, 1998). Both Bab1 and Bab2 are localized to the nucleus (Couderc et al., 2002; Godt and Laski, 1995; Godt et al., 1993) and interact with BIP2/ TAFII155, a component of TFIID (Pointud et al., 2001). Bab2 was also shown to bind at discrete sites along polytene chromosomes (D. Godt, unpubl. data; Pointud et al., 2001), suggesting that Bab1 and Bab2 function as transcriptional regulators. To conduct structure/function analyses of the two Bab proteins, including the examination of any functional differences between Bab1 and Bab2, we decided to develop a genetic assay that depends on the ability to rescue the bab mutant phenotype. However, the large size of the bab locus makes it unfeasible to generate a genomic rescue construct. Using bab cDNAs for rescue is an option, but bab has a complex expression pattern and initial attempts to isolate tissue-specific enhancers for driving bab cDNA expression that accurately reflect the normal bab expression pattern were not successful. We have expressed bab cDNAs with several promoters, such as the hsp70 heat shock promoter, but find that misexpression of the Bab proteins leads to cell death in many tissues (see accompanying article), making this approach less than ideal for studying bab function. A more amenable approach is to obtain a P[Gal4] enhancer trap line insertion (Brand, 1994; Brand and Perrimon, 1993) in the bab locus that expresses Gal4 in a bab-specific pattern allowing the use of UAS-bab constructs to express bab in its endogenous pattern. We used P element replacement to make bab P[Gal4] enhancer trap lines. This method is based on the finding that after a P element excises from a chromosome, a P element from a second site frequently transposes into the site of the excised P element (Gonzy-Treboul et al., 1995; Keeler and Gloor, 1997; Sepp and Auld, 1999). bab and bab are well characterized bab enhancer trap lines that express -gal in patterns very similar to the endogenous bab expression patterns (Couderc et al., 2002; Godt et al., 1993; Godt and Laski, 1995). bab is located within the first intron of the bab1 gene, whereas bab is located in the 5 untranslated region of the bab1 transcription unit. Here we describe the replacement of the P[ry , lacZ] insert in bab and bab with P[Gal4, w ] and show the Gal4 expression pattern of the resulting lines.

Here we describe how to generate customized microinjection needles from glass capillary tubes. Controls demonstrate the range of variables and effects on needle tip shape using a standard Flaming/Brown micropipette needle puller. Needles generated with two-cycle pulls provide a wider range of needle shapes in a predictable fashion. We used the needle puller’s ramp function for multiple-cycle programs to determine the useful range of heat settings inherent to the glass capillary tube. This article focuses primarily on the preparation of injection needles utilized for P-element-mediated germ-line transformation in Drosophila melanogaster that do not require the dechorionation of the egg. However, these types of needles can be useful for numerous other types of injections, such as RNA interference, homologous recombination mutagenesis, morpholinos, transient gene regulation, drug delivery, and the transfer of cytoplasmic factors that are useful in a wide range of biological systems ranging from plants to vertebrates. Using our standard needle, we correlate the survival of injected D. melanogaster embryos with transformation efficiencies and plasmid construct characteristics.

With the available eukaryotic genome sequences, there are predictions of thousands of previously uncharacterized genes without known function or available mutational variant. Thus, there is an urgent need for efficient genetic tools for genomewide phenotypic analysis. Here we describe such a tool: a deletion-generator technology that exploits properties of a double transposable element to produce molecularly defined deletions at high density and with high efficiency. This double element, called P { wHy }, is composed of a “deleter” element hobo , bracketed by two genetic markers and inserted into a “carrier” P element. We have used this P { wHy } element in Drosophila melanogaster to generate sets of nested deletions of sufficient coverage to discriminate among every transcription unit within 60 kb of the starting insertion site. Because these two types of mobile elements, carrier and deleter, can be found in other species, our strategy should be applicable to phenotypic analysis in a variety of model organisms.

Maintenance of proper neuronal excitability is vital to nervous system function and normal behavior. A subset of Drosophila mutants that exhibit altered behavior also exhibit defective motor neuron excitability, which can be monitored with electrophysiological methods. One such mutant is the P-element insertion mutant bemused (bem). The bem mutant exhibits female sterility, sluggishness, and increased motor neuron excitability. The bem P element is located in the large intron of the previously characterized translational repressor gene pumilio (pum). Here, by several criteria, we show that bem is a new allele of pum. First, ovary-specific expression of pum partially rescues bem female sterility. Second, pum null mutations fail to complement bem female sterility, behavioral defects, and neuronal hyperexcitability. Third, heads from bem mutant flies exhibit greatly reduced levels of Pum protein and the absence of two pum transcripts. Fourth, two previously identified pum mutants exhibit neuronal hyperexcitability. Fifth, overexpression of pum in the nervous system reduces neuronal excitability, which is the opposite phenotype to pum loss of function. Collectively, these findings describe a new role of pum in the regulation of neuronal excitability and may afford the opportunity to study the role of translational regulation in the maintenance of proper neuronal excitability.

Insulator DNAs and promoter competition regulate enhancer–promoter interactions within complex genetic loci. Here we provide evidence for a third mechanism: promoter-proximal tethering elements. The Scr - ftz region of the Antennapedia gene complex includes two known enhancers, AE1 and T1. AE1 selectively interacts with the ftz promoter to maintain pair-rule stripes of ftz expression during gastrulation and germ-band elongation. The T1 enhancer, located 3′ of the ftz gene and ≈25 kb 5′ of the Scr promoter, selectively activates Scr expression in the prothorax and posterior head segments. A variety of P element minigenes were examined in transgenic embryos to determine the basis for specific AE1- ftz and T1- Scr interactions. A 450-bp DNA fragment located ≈100 bp 5′ of the Scr transcription start site is essential for T1- Scr interactions and can mediate long-range activation of a ftz / lacZ reporter gene when placed 5′ of the ftz promoter. We suggest that the Scr 450 fragment contains tethering elements that selectively recruit T1 to the Scr promoter. Tethering elements might regulate enhancer–promoter interactions at other complex genetic loci.

Maternal transmission of RNAs or proteins through the egg cytoplasm plays an important role in eukaryotic development. We show that the transposase activity encoded by the P transposable element of Drosophila melanogaster is transmitted through the oocytes of females heterozygous for this element even when these oocytes do not carry the element itself. However, this maternal transmission is abolished when the last of three introns is removed from the P element. These facts imply that maternal transmission of transposase activity involves the RNA transcribed from the P element rather than the polypeptide it encodes, and that to be transmitted maternally, this RNA must possess the last intron. Examination of the intron's sequence reveals that it contains a motif of nine nucleotides that has been implicated in the maternal transmission of developmentally significant RNAs. This same intron limits expression of the P transposase to the germ line of Drosophila . Thus, the last P intron has two important biological functions.

The two haloacetate dehalogenase genes, dehH1 and dehH2 , on the 65-kb plasmid pUO1 from Delftia acidovorans strain B were found to be located on transposable elements. The dehH2 gene was carried on an 8.9-kb class I composite transposon (Tn Had1 ) that was flanked by two directly repeated copies of IS 1071 , IS 1071 L and IS 1071 R. The dehH1 gene was also flanked by IS 1071 L and a truncated version of IS 1071 (IS 1071 N). Tn Had1 , dehH1 , and IS 1071 N were located on a 15.6-kb class II transposon (Tn Had2 ) whose terminal inverted repeats and res site showed high homology with those of the Tn 21 -related transposons. Tn Had2 was defective in transposition because of its lacking the transposase and resolvase genes. Tn Had2 could transpose when the Tn 21 -encoded transposase and resolvase were supplied in trans . These results demonstrated that Tn Had2 is a defective Tn 21 -related transposon carrying another class I catabolic transposon.

Translocation of Sleeping Beauty (SB) transposon requires specific binding of SB transposase to inverted terminal repeats (ITRs) of about 230 bp at each end of the transposon, which is followed by a cut-and-paste transfer of the transposon into a target DNA sequence. The ITRs contain two imperfect direct repeats (DRs) of about 32 bp. The outer DRs are at the extreme ends of the transposon whereas the inner DRs are located inside the transposon, 165-166 bp from the outer DRs. Here we investigated the roles of the DR elements in transposition. Although there is a core transposase-binding sequence common to all of the DRs, additional adjacent sequences are required for transposition and these sequences vary in the different DRs. As a result, SB transposase binds less tightly to the outer DRs than to the inner DRs. Two DRs are required in each ITR for transposition but they are not interchangeable for efficient transposition. Each DR appears to have a distinctive role in transposition. The spacing and sequence between the DR elements in an ITR affect transposition rates, suggesting a constrained geometry is involved in the interactions of SB transposase molecules in order to achieve precise mobilization. Transposons are flanked by TA dinucleotide base-pairs that are important for excision; elimination of the TA motif on one side of the transposon significantly reduces transposition while loss of TAs on both flanks of the transposon abolishes transposition. These findings have led to the construction of a more advanced transposon that should be useful in gene transfer and insertional mutagenesis in vertebrates. (C) 2002 Elsevier Science Ltd. All rights reserved.

A new way was developed to isolate rhythmically expressed genes in Drosophila by modifying the classic enhancer-trap method. We constructed a P element containing sequences that encode firefly luciferase as a reporter for oscillating gene expression in live flies. After generation of 1176 autosomal insertion lines, bioluminescence screening revealed rhythmic reporter-gene activity in 6% of these strains. Rhythmically fluctuating reporter levels were shown to be altered by clock mutations in genes that specify various circadian transcription factors or repressors. Intriguingly, rhythmic luminescence in certain lines was affected by only a subset of the pacemaker mutations. By isolating genes near 13 of the transposon insertions and determining their temporal mRNA expression pattern, we found that four of the loci adjacent to the trapped enhancers are rhythmically expressed. Therefore, this approach is suitable for identifying genetic loci regulated by the circadian clock. One transposon insert caused a mutation in the rhythmically expressed gene numb. This novel numb allele, as well as previously described ones, was shown to affect the fly's rhythm of locomotor activity. In addition to its known role in cell fate determination, this gene and the phosphotyrosine-binding protein it encodes are likely to function in the circadian system.

Cuticular pheromones in Drosophila melanogaster are unsaturated hydrocarbons with at least one double bond in position 7: 7-tricosene and 7-pentacosene in males and 7,11 -heptacosadiene and 7,11 -nonacosadiene in females. We have previously shown that a desaturase gene, desat1, located in chromosome region 87 C could be involved in this process: the Desat1 enzyme preferentially leads to the synthesis of palmitoleic acid, a precursor of omega7 fatty acids and 7-unsaturated hydrocarbons. Therefore, we have searched for P-elements in the 87 region and mapped them. One was found inserted into the first intron of the desat1 gene. Flies heterozygous for this insertion showed a large decrease in the level of 7-unsaturated hydrocarbons, comparable to that observed in flies heterozygous for a deficiency overlapping desat1. Less than 1 % of flies homozygous for this insertion were viable. They were characterized by dramatic pheromone decreases. After excision of the transposon, the pheromone phenotype was reversed in 69% of the lines and the other excision lines had more or less decreased amounts of 7-unsaturated hydrocarbons. All these results implicate desat1 in the synthesis of Drosophila pheromones.

The Drosophila melanogaster genome consists of four chromosomes that contain 165 Mb of DNA, 120 Mb of which are euchromatic. The two Drosophila Genome Projects, in collaboration with Celera Genomics Systems, have sequenced the genome, complementing the previously established physical and genetic maps. In addition, the Berkeley Drosophila Genome Project has undertaken large-scale functional analysis based on mutagenesis by transposable P element insertions into autosomes. Here, we present a large-scale P element insertion screen for vital gene functions and a BAC tiling map for the X chromosome. A collection of 501 X-chromosomal P element insertion lines was used to map essential genes cytogenetically and to establish short sequence tags (STSs) linking the insertion sites to the genome. The distribution of the P element integration sites, the identified genes and transcription units as well as the expression patterns of the P-element-tagged enhancers is described and discussed.

Many vectors are available for expression of cloned genes in the Drosophila germ line and eye. Nevertheless, for experiments that require female germ line expression, it is often problematic to find a vector that directs transcription suitably early and at high enough levels. In addition, a vector specific for undifferentiated cells in the Drosophila eye has yet to be described. Here, we have used the enhancer and promoter sequences of the fat facets gene to construct a P element transformation vector, pFAF, that provides high levels of gene expression in the female germ line. We present evidence that pFAF is a more potent activator of transcription in the female germ line than other commonly used vectors. In addition, in the eye, pFAF is unique in that it activates transcription specifically in undifferentiated cells.

Transgene insertion plays an important role in gene therapy and in biological studies. Transposon-based systems that integrate transgenes by transposase-catalyzed “cut-and-paste” mechanism have emerged as an attractive system for transgenesis. Hyperactive piggyBac transposon is particularly promising due to its ability to integrate large transgenes with high efficiency. However, prolonged expression of transposase can become a potential source of genotoxic effects due to uncontrolled transposition of the integrated transgene from one chromosomal locus to another. In this study we propose a vector design to decrease post-transposition expression of transposase and to eliminate the cells that have residual transposase expression. We design a single plasmid construct that combines the transposase and the transpositioning transgene element to share a single polyA sequence for termination. Consequently, the separation of the transposase element from the polyA sequence after transposition leads to its deactivation. We also co-express Herpes Simplex Virus thymidine kinase (HSV-tk) with the transposase. Therefore, cells having residual transposase expression can be eliminated by the administration of ganciclovir. We demonstrate the utility of this combination transposon system by integrating and expressing a model therapeutic gene, human coagulation Factor IX, in HEK293T cells.

The ability to genetically transform and modify Drosophila melanogaster was originally made possible through the use of transposable elements (TEs). These proved to be efficient mutagens leading to the generation of many libraries containing TE-tagged genes and enhancers throughout much of the genome. TEs were also efficient at introducing genes into this insect. A limitation of this technology was the inability to direct where transposons insert in the genome. However, recent advances made with the development of targeting systems based on protein-based and RNA-based guidance of endonucleases to specific target sites have provided a solution to this problem. As a consequence, the genetic toolbox of Drosophila geneticists have considerably expanded and will have a dramatic impact on our ability to understand genetic pathways in this insect. Key Concepts: Class II transposable elements form the basis of versatile genetic technology. Homologous recombination technologies have been problematic but progress has been made. Site-specific recombinases add an additional dimension to the genetic toolbox. Homologous recombination technologies have been problematic but progress has been made. Zinc finger nucleases provide an alternate approach to site-specificity. TALENs facilitate another approach to directed integration into the genome. CRISPRs will lead to a new generation of approaches to site-specific medication in Drosophila. Keywords: transposable element; homologous recombination; zinc finger nucleases; TALENs; CRISPR/Cas9

A P-element line ( P0997) of Drosophila melanogaster in which the P element disrupts the Drosophila homolog of the Saccharomyces cerevisiae gene APG4/AUT2 was identified during the course of screening for cut ( ct) modifiers. The yeast gene APG4/AUT2 encodes a cysteine endoprotease directed against Apg8/Aut7 and is necessary for autophagy. The P0997 mutation enhances the wing margin loss associated with ct mutations, and also modifies the wing and eye phenotypes of Notch (N), Serrate (Ser), Delta (Dl), Hairless (H), deltex (dx), vestigial (vg) and strawberry notch (sno) mutants. These results therefore suggest an unexpected link between autophagy and the Notch signaling pathway.

In Drosophila, clusters of P transgenes (P-lac-w) display a variegating phenotype for the w marker. In addition, X-ray-induced rearrangements of chromosomes bearing such clusters may lead to enhancement of the variegated phenotype. Since P-lacZ transgenes in subtelomeric heterochromatin have some P-element repression abilities, we tested whether P-lac-w clusters also have the capacity to repress P-element activity in the germline. One cluster (T-1), located on a rearranged chromosome (T2;3) and derived from a line bearing a variegating tandem array of seven P-lac-w elements, partially represses the dysgenic sterility (GD sterility) induced by P elements. This cluster also strongly represses in trans the expression of P-lacZ elements in the germline. This latter suppression shows a maternal effect. Finally, the combination of variegating P-lac-w clusters and a single P-lacZ reporter inserted in subtelomeric heterochromatic sequences at the X chromosome telomere (cytological site 1A) leads to strong repression of dysgenic sterility. These results show that repression of P-induced dysgenic sterility can be elicited in the absence of P elements encoding a polypeptide repressor and that a transgene cluster can repress the expression of a single homologous transgene at a nonallelic position. Implications for models of transposable element silencing are discussed.

Transgenic non‐Drosophilid insects have been made using insect transposable elements that have a broad host range such as the piggyBac element. However, the success rate is often low. Previous piggyBac helper plasmids have used either the piggyBac or the hsp70 promoter from Drosophila melanogaster to control expression of the transposase gene. Here we show that plasmids with the piggyBac transposase gene regulated by constitutive promoters can be effective ‘helpers’ for mediating transposition in D. melanogaster. We also present preliminary evidence on the use of an RNA helper that encodes the transposase. Our results suggest that for germ‐line transformation of non‐Drosophilid insects it may be advantageous to isolate a constitutive promoter from the species of interest to control transposase expression.

Transposable elements have been used widely in the past 20 years for gene transfer and insertional mutagenesis in Drosophila . Transposon-based technology for gene manipulation and genomic analysis currently is being adopted for vertebrates. We tested the ability of Minos , a DNA transposon from Drosophila hydei , to transpose in mouse tissues. Two transgenic mouse lines were crossed, one expressing Minos transposase in lymphocytes under the control of the CD2 promoter/locus control region and another carrying a nonautonomous Minos transposon. Only mice containing both transgenes show excision of the transposon and transposition into new chromosomal sites in thymus and spleen cells. In addition, expression of Minos transposase in embryonic fibroblast cell lines derived from a transposon-carrying transgenic mouse resulted in excision of the transposon. These results are a first step toward a reversible insertional mutagenesis system in the mouse, opening the way to develop powerful technologies for functional genomic analysis in mammals.

Sequences homologous to the P element of Drosophila melanogaster were previously identified in Drosophila mediopunctata, a member of the tripunctata group, subgenus Drosophila. We report here that the P element is present in about three to five copies in the D. mediopunctata genome. While one of the insertion sites appears to be fixed, others may be polymorphic, indicating relatively recent P element activity. Phylogenetic analysis revealed that the D. mediopunctata element belongs to the canonical subfamily of P elements and that divergence of the D. mediopunctata element from other members of this subfamily ranges from 2% to 5% at the nucleotide level. This is the first report of a canonical P element outside the subgenus Sophophora. Based primarily on the striking incongruence between P element and host species phylogenies, the presence of a canonical P element in D. mediopunctata is most likely explained by horizontal transfer between species.

P element somatic inhibitor (PSI) is a 97-kDa RNA-binding protein with four KH motifs that is involved in the inhibition of splicing of the Drosophila P element third intron (IVS3) in somatic cells. PSI interacts with a negative regulatory element in the IVS3 5' exon. This element contains two pseudo-5' splice sites, termed F1 and F2. To identify high affinity binding sites for the PSI protein, in vitro selection (SELEX) was performed using a random RNA oligonucleotide pool. Alignment of high affinity PSI-binding RNAs revealed a degenerate consensus sequence consisting of a short core motif of CUU flanked by alternative purines and pyrimidines. Interestingly, this sequence resembles the F2 pseudo-5' splice site in the P element negative regulatory element. Additionally, a negative in vitro selection of PCR-mutagenized P element 5' exon regulatory element RNAs identified two U residues in the F1 and F2 pseudo-5' splice sites as important nucleotides for PSI binding and the U residue in the F2 region is a nearly invariant nucleotide in the consensus SELEX motif. The high affinity PSI SELEX sequence acted as a splicing inhibitor when placed in the context of a P element splicing pre-mRNA in vitro. Data from in vitro splicing assays, UV crosslinking and RNA-binding competition experiments indicates a strong correlation between the binding affinities of PSI for the SELEX sequences and their ability to modulate splicing of P element IVS3 in vitro.

In order to study P element dynamics in natural populations of Drosophila melanogaster, 126 isofemale lines were examined from seven of the southernmost islands of Japan (the Sakishima Islands) and from Taiwan. Gonadal dysgenesis (GD) tests showed large divergences in the P-M phenotypes (P inducing and P repressing abilities) between the island populations. The P-M characteristics of each population, however, had not greatly changed in the past 15 years. Their genomic P element profiles are highly similar, consisting mostly of full-size P and of KP elements. We found no clear relationship between phenotype and genomic P element composition.

We have further characterized essential loci within the centric heterochromatin of the left arm of chromosome 3 (3L) of Drosophila melanogaster, using EMS, radiation and P element mutagenesis. We failed to find any new essential genes, a result that suggests a lower-than-average gene density in this region. Mutations affecting expression of the most proximal gene [lethal 1, l1 or l(3)80Fj] act as dominant suppressors of Polycomb (Pc), behavior which is consistent with a putative trithorax group (trx-G) gene. The third gene to the left of the centromere [lethal 3, l3 or l(3)80Fh] is likely to correspond to verthandi (vtd), a known trx-G gene that plays a role in the regulation of hedgehog (hh) expression and signalling. The intervening gene [lethal 2, l2 or l(3)80Fi] is required throughout development, and mutant alleles have interesting phenotypes; in various allelic combinations that survive, we observe fertility, bristle, wing, eye and cuticle defects.

In Drosophila melanogaster, gain-of-function mutagenesis utilizing the GAL4-UAS system has been established, allowing identification of genes that may not be easily detectable by loss-of-function screening approaches. The conditional features of misexpression systems are especially useful for studying late-stage biological processes, such as those involving adult behavior or lifespan. The gene search system, incorporating a bidirectional misexpression vector, was used to screen for genes critical for longevity determination. We have identified several genes whose misexpression in adulthood extends the fly's lifespan. Phenotypic characterization of fly lines carrying a mis-expression vector, in conjunction with obtaining information about the genomic insertion sites, creates valuable resources for the systematic functional genomics in Drosophila.

A novel tagging system AcREH, designed for obtaining gain-of-function mutations, was prepared on the basis of a self-stabilizing Ac transposon derivative. The transposable element, DsAT, was constructed in a way that it can activate transcription of neighboring genes by two 35S promoters and/or by four tandem repeats of the enhancer fragment of this promoter. DsAT revealed somatic excision in the first generation of the tobacco transformants. The element exhibited germinal excision to the next generation, as demonstrated by PCR and Southern hybridization analysis. In spite of the structure of the element, which may inhibit the expression of the transposase gene, the frequency of germinal excision was comparable to or higher than those so far reported, suggesting the applicability of the element for gene tagging.

Hybrid dysgenesis provides a brilliant example of ME mobilization, which causes various mutations and other genetic alterations, including gonadal sterility [5, 6]. Three independent systems of hybrid dysgenesis have been described to date in D. melanogaster, and the above alterations result in each case from activa tion of one ME: P element, I element, or hobo [5–7]. On the other hand, at least six unrelated MEs are mobilized in hybrid dysgenesis in D. virilis [8, 9]. A key role in their mobilization is ascribed to Penelope [10, 11].

Aging is genetically determined and environmentally modulated. In a study of longevity in the adult fruit fly, Drosophila melanogaster , we found that five independent P-element insertional mutations in a single gene resulted in a near doubling of the average adult life-span without a decline in fertility or physical activity. Sequence analysis revealed that the product of this gene, named Indy (for I'm not dead yet ), is most closely related to a mammalian sodium dicarboxylate cotransporter—a membrane protein that transports Krebs cycle intermediates. Indy was most abundantly expressed in the fat body, midgut, and oenocytes: the principal sites of intermediary metabolism in the fly. Excision of the P element resulted in a reversion to normal life-span. These mutations may create a metabolic state that mimics caloric restriction, which has been shown to extend life-span.

P elements of two different subfamilies designated as M- and O-type are thought to have invaded host species in the Drosophila obscura group via horizontal transmission from external sources. Sequence comparisons with P elements isolated from other species suggested that the horizontal invasion by the O-type must have been a rather recent event, whereas the M-type invasion should have occurred in the more distant past. To trace the phylogenetic history of O-type elements, additional taxa were screened for the presence of O- and M-type elements using type-specific PCR primers. The phylogeny deduced from the sequence data of a 927-bp section (14 taxa) indicate that O-type elements have undergone longer periods of regular vertical transmission in the lineages of the saltans and willistoni groups of Drosophila. However, starting from a species of the D. willistoni group they were transmitted horizontally into other lineages. First the lineage of the D. affinis subgroup was infected, and finally, in a more recent wave of horizontal spread, species of three different genera were invaded by O-type elements from the D. affinis lineage: Scaptomyza, Lordiphosa, and the sibling species D. bifasciata/D. imaii of the Drosophila obscura subgroup. The O-type elements isolated from these taxa are almost identical (sequence divergence

An ethylmethane sulfonate (EMS) mutagenesis of Drosophila melanogaster aimed at discovering novel genes essential for neuromuscular development identified six embryonic lethal alleles of one genetic locus on the third chromosome at 62C. Two additional lethal P element insertion lines, l(3)S02001 and l(3)j1D7, failed to complement each other and each of the six EMS alleles. Analysis of genomic sequence bracketing the two insertion sites predicted a protein of 16,215 amino acid residues, encoded by a 70 kb genomic region. This sequence includes the recently characterized kettin, and includes all known partial D-Titin sequences. We call the genetic locus, which encodes both D-Titin and kettin, D-Titin. D-Titin has 53 repeats of the immunoglobulin C2 domain, 6 repeats of the fibronectin type III domain and two large PEVK domains. Kettin appears to be the NH2-terminal one third of D-Titin, presumably expressed via alternative splicing. Phenotype assays on the allelic series of D-Titin mutants demonstrated that D-Titin plays an essential role in muscle development. First, D-Titin has an unsuspected function in myoblast fusion during myogenesis and, second, D-Titin later serves to organize myofilaments into the highly ordered arrays underlying skeletal muscle striation. We propose that D-Titin is instrumental in the development of the two defining features of striated muscle: the formation of multi-nucleate syncitia and the organization of actin-myosin filaments into striated arrays.

Microsomal glutathione S-transferase-I (MGST-I) has been thought to be important for protecting the cell from oxidative damages and/or xenobiotics. We have previously identified the Microsomal glutathione S-transferase-like (Mgstl) gene, a Drosophila homologue of human MGST-I. To investigate the function of the enzyme using Drosophila as a model system, we examined the expression pattern of Mgstl during development, and generated loss-of-function mutants to assess its in-vivo function. Mgstl was expressed in all developmental stages. It is expressed ubiquitously with the highest expression in the larval fat body, an insect organ thought to be functionally corresponding to mammalian liver, while relatively low in the central nervous system. This tissue distribution is consistent with that of MGST-I in humans or Rats. Mgstl null mutants generated from a P element insertion line showed no obvious defects in morphology, indicating that it is not essential for the development. However, their life span was significantly reduced compared to control flies, suggesting that the MGSTL protein is involved in processes somehow contributing to aging. We found an Mgstl pseudogene, which is apparently derived through the reverse transcription of Mgstl mRNA and subsequent integration into the genome.

In Drosophila melanogaster, hybrid dysgenesis occurs in progeny from crosses between females lacking P elements and males carrying P elements scattered throughout the genome. We have genetically isolated a naturally occurring P insertion at cytological location 1A, from a Tunisian population. The Nasr'Allah-P(1A) element [NA-P(1A)] has a deletion of the first 871 bp including the P promoter. It is flanked at the 3′ end by telomeric associated sequences and at the 5′ end by a HeT-A element sequence. The NA-P(1A) element strongly represses dysgenic sterility and P transposition. However, when testing P-promoter repression, NA-P(1A) was unable to repress a germinally expressed P-lacZ construct bearing no 5′-homology with it. Conversely, a second P-lacZ construct, in which the fusion with lacZ takes place in exon 3 of P, was successfully repressed by NA-P(1A). This suggests that NA-P(1A) repression involves a homology-dependent component.

The courtless (col) mutation disrupts early steps of courtship behavior in Drosophila males, as well as the development of their sperm. Most of the homozygous col/col males (78%) do not court at all. Only 5% perform the entire ritual and copulate, yet these matings produce no progeny. The col gene maps to polytene chromosome band 47D. It encodes two proteins that differ in their carboxy termini and are the Drosophila homologs of the yeast ubiquitin-conjugating enzyme UBC7. The col mutation is caused by an insertion of a P element into the 3′ UTR of the gene, which probably disrupts translational regulatory elements. As a consequence, the homozygous mutants exhibit a six- to sevenfold increase in the level of the COL protein. The col product is essential, and deletions that remove the col gene are lethal. During embryonic development col is expressed primarily in the CNS. Our results implicate the ubiquitin-mediated system in the development and function of the nervous system and in meiosis during spermatogenesis.

The recent completion of the Drosophila genome sequence opens new avenues for neurobiology research. We screened the fly genome sequence for homologs of mammalian genes implicated directly or indirectly in exocytosis and endocytosis of synaptic vesicles. We identified fly homologs for 93% of the vertebrate genes that were screened. These are on average 60% identical and 74% similar to their vertebrate counterparts. This high degree of conservation suggests that little protein diversification has been tolerated in the evolution of synaptic transmission. Finally, and perhaps most exciting for Drosophila neurobiologists, the genomic sequence allows us to identify P element transposon insertions in or near genes, thereby allowing rapid isolation of mutations in genes of interest. Analysis of the phenotypes of these mutants should accelerate our understanding of the role of numerous proteins implicated in synaptic transmission.