Element-mediated Transformation Research Articles

The Unusual Transposable Element Penelope and Its Behavior in Distant Drosophila Species

The retroelement Penelope isolated from Drosophila virilis has a very unusual structure and codes for reverse transcriptase and an endonuclease belonging to the UvrC type. As shown previously,Penelope is a key element in induction of the hybrid dysgenesis syndrome described in D. virilis, which also involves mobilization of several unrelated mobile element families. Here we report a successful introduction of Penelope into the D. melanogaster genome by P element-mediated transformation. In the new host genome,Penelope is actively transcribed producing major transcript which coincides with that detected in dysgenic hybrids of D. virilis. In situ hybridization on D. melanogaster polytene chromosomes and Southern blotting revealed multiple transpositions of Penelopein the transformed D. melanogaster strains. We determined the structure of six Penelope copies inserted into D. melanogaster chromosomes. Some transformed D. melanogaster strains showed dysgenesis effects similar to those observed in hybrids from D. virilis dysgenic crosses.

Penelope retroelements from Drosophila virilis are active after transformation of Drosophila melanogaster.

The Penelope family of retroelements was first described in species of the Drosophila virilis group. Intact elements encode a reverse transcriptase and an endonuclease of the UvrC type, which may play a role in Penelope integration. Penelope is a key element in the induction of D. virilis hybrid dysgenesis, which involves the mobilization of several unrelated families of transposable elements. We here report the successful introduction of Penelope into the germ line of Drosophila melanogaster by P element-mediated transformation with three different constructs. Penelope is actively transcribed in the D. melanogaster genome only in lines transformed with a construct containing a full-length Penelope clone. The transcript is identical to that detected in D. virilis dysgenic hybrids. Most newly transposed Penelope elements have a very complex organization. Significant proliferation of Penelope copy number occurred in some lines during the 24-month period after transformation. The absence of copy number increase with two other constructs suggests that the 5' andor 3' UTRs of Penelope are required for successful transposition in D. melanogaster. No insect retroelement has previously been reported to be actively transcribed and to increase in copy number after interspecific transformation.

Analysis of core promoter sequences located downstream from the TATA element in the hsp70 promoter from Drosophila melanogaster.

TFIID recognizes multiple sequence elements in the hsp70 promoter of Drosophila. Here, we investigate the function of sequences downstream from the TATA element. A mutation in the initiator was identified that caused an eightfold reduction in binding of TFIID and a fourfold reduction in transcription in vitro. Another mutation in the +24 to +29 region was somewhat less inhibitory, but a mutation in the +14 to +19 region had essentially no effect. The normal promoter and the mutants in the initiator and the +24 to +29 region were transformed into flies by P element-mediated transformation. The initiator mutation reduced expression an average of twofold in adult flies, whereas the mutation in the +24 to +29 region had essentially no effect. In contrast, a promoter combining the two mutations was expressed an average of sixfold less than the wild type. The results suggest that the initiator and the +24 to +29 region could serve overlapping functions in vivo. Protein-DNA cross-linking was used to identify which subunits of TFIID contact the +24 to +29 region and the initiator. No specific subunits were found to cross-link to the +24 to +29 region. In contrast, the initiator cross-linked exclusively to dTAF230. Remarkably, dTAF230 cross-links approximately 10 times more efficiently to the nontranscribed strand than to the transcribed strand at the initiator.

Controlled expression of tagged proteins in Drosophila using a new modular P-element vector system.

We have developed a new modular vector system that facilitates the combination of various DNA fragments as functional modules for P element-mediated transformation of Drosophila melanogaster. The basic vector pP¿GS¿ contains unique sites for 17 restriction enzymes, including three 8-bp cutters, that allow one to combine various promoter elements, cDNAs and genomic DNA fragments, as well as protein tags and selectable marker genes, for a wide spectrum of transgene analyses. With this new vector system we analysed the chromosomal distribution of the Drosophila SU(VAR)3-9 protein tagged with EGFP, using hsp70-cDNA and genomic Su(var)3-9 constructs. We found preferential association of the tagged SU(VAR)3-9 with centric heterochromatin.

Transgenes in the Analysis of Life Span and Fitness.

Drosophila P element-mediated transformation can be used to determine whether and how a specific gene contributes to demographic components of fitness. Motivated by the problem of senescence, researchers have applied this approach to genes thought to affect survival through processes of somatic maintenance. Cu/Zn-superoxide dismutase and catalase reduce the flux of reactive oxygen molecules that are thought to be a central cause of aging. EF1α is a component of the protein synthesis machine; deterioration of this housekeeping function is a potential contributor to senescence. Molecular chaperones such as the heat shock protein hsp70 are multifunctional molecules that affect a cell's response to acute stress. In some models, senescence results from the cumulative effects of stress, and heat shock proteins may regulate the progress of this deterioration. Transformations with the candidate genes of these proteins were used in independent studies to measure the effect of overexpression on longevity; positive results were reported. Here, I discuss the robustness of these results. I use the studies of superoxide dismutase, catalase, and EF1α to illustrate how the mutagenic effects of inserts confound our interpretations. I present new data from a reported study of hsp70 overexpression to show how engineered constructs can be used to overcome mutagenic artifacts through the controlled excision of sequences or alleles. The data for hsp70 provide the first strong molecular evidence that somatic maintenance affects longevity. Finally, future potential uses of transformation with Drosophila are discussed. I consider how metabolic control theory predicts that overexpression of genes for enzymes of intermediary metabolism is not likely to produce analytically useful changes in components of fitness.

E2F mediates developmental and cell cycle regulation of ORC1 in Drosophila.

Throughout the cell cycle of Saccharomyces cerevisiae, the level of origin recognition complex (ORC) is constant and ORCs are bound constitutively to replication origins. Replication is regulated by the recruitment of additional factors such as CDC6. ORC components are widely conserved, and it generally has been assumed that they are also stable factors bound to origins throughout the cell cycle. In this report, we show that the level of the ORC1 subunit changes dramatically throughout Drosophila development. The accumulation of ORC1 is regulated by E2F-dependent transcription. In embryos, ORC1 accumulates preferentially in proliferating cells. In the eye imaginal disc, ORC1 accumulation is cell cycle regulated, with high levels in late G1 and S phase. In the ovary, the sub-nuclear distribution of ORC1 shifts during a developmentally regulated switch from endoreplication of the entire genome to amplification of the chorion gene clusters. Furthermore, we find that overexpression of ORC1 alters the pattern of DNA synthesis in the eye disc and the ovary. Thus, replication origin activity appears to be governed in part by the level of ORC1 in Drosophila.

Regulation of the expression of the sn-glycerol-3-phosphate dehydrogenase gene in Drosophila melanogaster.

P element-mediated transformation has been used to investigate the regulation of expression of the sn-glycerol-3-phosphate dehydrogenase gene of Drosophila melanogaster. A 13-kb construct containing the eight exons and associated introns, 5 kb of the 5' region, and 3 kb downstream from the structural gene produced normal levels of enzyme activity and rescued the poor viability of flies lacking the enzyme. All the regulatory elements essential for normal enzyme expression were located in a fragment that included the exons and introns and 1-kb upstream noncoding sequence. Deletions of the 1.6-kb second intron reduced activity to 25%. Transformants with fusion constructs between the sn-glycerol-3-phosphate dehydrogenase gene and the beta-galactosidase gene from E. coli revealed three elements that affected expression. A (CT)9 repeat element at the 5' end of the second intron increased expression in both larvae and adults, particularly at emergence. A second regulatory element, which includes a (CT)7 repeat, was located 5' to the TATA box and had similar effects on the gene's expression. A third, undefined, enhancer was located in the second intron, between 0.5 and 1.8 kb downstream of the translation initiation codon. This element increases enzyme activity to a similar extent in larvae and adults but has little effect when the enhancer at the 5' end of the intron is present.

FLP-mediated DNA mobilization to specific target sites in Drosophila chromosomes.

The ability to place a series of gene constructs at a specific site in the genome opens new possibilities for the experimental examination of gene expression and chromosomal position effects. We report that the FLP- FRT site-specific recombination system of the yeast 2mu plasmid can be used to integrate DNA at a chromosomal FRT target site in Drosophila. The technique we used was to first integrate an FRT- flanked gene by standard P element-mediated transformation. FLP was then used to excise the FRT- flanked donor DNA and screen for FLP-mediated re-integration at an FRT target at a different chromosome location. Such events were recovered from up to 5% of the crosses used to screen for mobilization and are easily detectable by altered linkage of a white reporter gene or by the generation of a white + gene upon integration.

Molecular Analysis of theDrosophilaCatalase Gene

The main objective of this study was to isolate and characterize the catalase gene and accompanyingcis-regulatory regions inDrosophila melanogaster.Genomic clones were obtained on the basis of cross-hybridization to catalase cDNA and a 7-kbSalI–KpnI fragment encompassing the catalase gene was introduced intoDrosophilaby P element-mediated transformation. A single transgene, when placed in a catalase null background, was sufficient to restore resistance to H2O2as well as reduce susceptibility to early death. DNA sequence of the catalase gene domain was obtained. This included 1365 bp of sequence upstream of the transcription initiation site and 1423 bp downstream of the termination codon. TheDrosophilacatalase gene is composed of 3 exons, encoding 19, 307, and 180 amino acids, which are separated by 3520- and 96-bp introns. Sequence analysis of the promoter domain is presented, revealing multiple sequence similarities between catalase and Cu,Zn superoxide dismutase promoter domains. Developmental RNA gel analysis shows that peaks of catalase mRNA accumulation correspond roughly with major peaks of ecdysone titer during third instar and pupal stages. Candidate ecdysone response element sequences are noted downstream of the catalase polyadenylation site.

Interactions between the regulatory regions of two Adh alleles.

A region (NS1) that acts like an enhancer is located approximately 300 bp upstream of the larval cap site in the Adh gene of D. melanogaster. When this sequence is deleted (delta NS1), the gene fails to express ADH protein. Gene expression can be restored by placing a second Adh gene with an intact enhancer elsewhere on the same plasmid. In these circumstances, both genes are expressed equally regardless of their orientation on the plasmid. In this report we further characterize the interactions that occur when a single enhancer activates expression from a proximal and distant promoter. We have made the following observations: (1) While the two genes are expressed equivalently, their expression relative to a plasmid carrying two intact genes is reduced by a factor of 2 to 6 depending on the orientation of the two genes. (2) The single enhancer drives expression of both genes on any given plasmid molecule. (3) The enhancer does not interact with the Adh gene from which the NS7 region (which spans the larval TATA box) is removed. (4) Expression of the delta NS1 gene can be restored by an intact gene when both are inserted together into the Drosophila genome via P element-mediated transformation. (5) Increasing the separation between the two genes on a plasmid by up to 15 kbp does not prevent the restoration of expression of the delta NS1 gene. We propose a model that explains how a single enhancer can stimulate equal expression from two genes.

Expression and targeting of Syrian hamster prion protein induced by heat shock in transgenic Drosophila melanogaster

To evaluate the fruit fly as a model for studying neurodegenerative diseases caused by prions, transgenic flies were generated by introducing the Syrian hamster prion protein (SHaPrP) gene into the Drosophila melanogaster germ line by P element-mediated transformation. Nine transgenic lines were isolated; induction of transgenes that had been placed under the control of the Drosophila heat shock promoter, hsp 70, resulted in the synthesis of full-length SHaPrP. The relative molecular weight of the recombinant protein was lower than that of authentic SHaPrP due to incomplete processing of Asn-linked CHOs. To determine the cellular localization of SHaPrP, Drosophila Schneider line 2 cells were transfected with the same constructs used for fly transformation. Heat shock induced SHaPrP was anchored to the surface of S2 cells by a glycolipid, demonstrating that the carboxy-terminal glycolipidation signal of SHaPrP is recognized by this evolutionarily distant host. When SHaPrP was synthesized in transgenic flies constitutively by subjecting them to heat pulses continuously, no difference in their lifespans compared with controls was detected. Furthermore, expression of SHaPrP for 20 days did not produce protease resistant SHaPrP, which is the major and possibly only component of the infectious prion. In contrast to transgenic mice overexpressing SHaPrP, which develop a profound neuromyopathy, no disease phenotype was associated with expression of SHaPrP over the entire lifespan of transgenic flies.

Influence of chromosomal position and copy number of a white-directed ribozyme gene on the suppression of eye pigmentation in Drosophila melanogaster.

Different strains of transgenic Drosophila melanogaster carrying one, two, or three copies of a heat-shock promoter 70 (hsp70)-driven catalytic antisense RNA gene, directed against the white gene, were investigated for the expression level of ribozyme RNA. It was found that the steady-state concentrations of the hammerhead ribozyme were proportional to the copy number of the genes and that the suppressive effect on eye pigment accumulation was dosage dependent. In a further experiment, a D. melanogaster strain, deficient in eye pigmentation caused by a deletion of the white gene, was used for P element-mediated germline transformation: the transposon used contained the hsp70-driven, white-directed ribozyme gene and, on the same DNA, the mini-white gene under its own promoter. The spatial coupling of the transcription of ribozyme and target RNA resulted in more effective ribozyme-mediated inhibition of eye pigmentation under heat-shock conditions. These effects were dependent on the chromosomal integration site of the transposon.

Mobile Minos elements from Drosophila hydei encode a two-exon transposase with similarity to the paired DNA-binding domain.

Elements related to the Tc1-like Minos mobile element have been cloned from Drosophila hydei and sequenced. Southern blot and sequence analyses show that (i) the elements are actively transposing in the Drosophila hydei germ line, (ii) they are characterized by a striking degree of sequence and size homogeneity, and (iii) like Tc1, they insert at a TA dinucleotide that is probably duplicated during the process. The nucleotide sequences of two elements, Minos-2 and Minos-3, differ at only one position from each other and contain two nonoverlapping open reading frames that are separated by a putative 60-nucleotide intron. The amino-terminal part of the Minos putative transposase shows sequence similarity to the paired DNA-binding domain. Forced transcription of a modified Minos element that was introduced into the Drosophila melanogaster germ line by P element-mediated transformation resulted in the production of accurately spliced polyadenylylated RNA molecules. It is proposed that Minos-2 and/or Minos-3 may encode an active transposase containing an amino-terminal DNA-binding domain that is distantly related to the paired DNA-binding domain.

The mago nashi locus encodes an essential product required for germ plasm assembly in Drosophila

In Drosophila, the localization of maternal determinants to the posterior pole of the oocyte is required for abdominal segmentation and germ cell formation. These processes are disrupted by maternal effect mutations in ten genes that constitute the posterior group. Here, the molecular analysis of one posterior group gene, mago nashi, is presented. Restriction fragment length polymorphisms and transcript alterations associated with mago nashi mutations were used to identify the mago nashi locus within a chromosomal walk. The mago nashi locus was sequenced and found to encode a 147 amino acid protein with no similarity to proteins of known or suspected function. The identification of the mago nashi locus was confirmed by sequencing mutant alleles and by P element-mediated transformation. Nonsense mutations in mago nashi, as well as a deletion of the 5' coding sequences, result in zygotic lethality. The original mago nashi allele disrupts the localization of oskar mRNA and staufen protein to the posterior pole of the oocyte during oogenesis; anterior localization of bicoid mRNA is unaffected by the mutation. These results demonstrate that mago nashi encodes an essential product necessary for the localization of germ plasm components to the posterior pole of the oocyte.

Expression of an amylase--alcohol dehydrogenase chimeric gene in transgenic strains of Drosophila melanogaster.

A chimeric gene, consisting of 428 bp of the promoter sequences of the alpha-amylase gene of Drosophila melanogaster, fused to the transcribed region of the alcohol dehydrogenase (Adh) gene, was introduced into the genome of an Adhnull stock of Drosophila via P element mediated transformation. DNA analysis (Southern blotting) of three transformant strains confirmed the insertion of either one or two copies of the chimeric gene per strain. A histochemical study of ADH enzyme activity in dissected tissues of the transgenic larvae revealed that the chimeric Amy-Adh gene was expressed only in the posterior larval midgut and that this expression was repressed by dietary glucose, thus representing an expression pattern characteristic of the Amy gene. This indicates that the Amy upstream promoter sequences contain signals mediating both tissue specificity and glucose repression of the Adh structural gene in the transgenic larvae. The level of ADH activity expressed in transgenic flies was relatively low. This was paralleled by a low level of Adh mRNA, indicating a reduction in the transcriptional rate of the chimeric gene.

The Drosophila stubarista phenotype is associated with a dosage effect of the putative ribosome-associated protein D-p40 on spineless.

We describe the molecular characterization of the Drosophila melanogaster gene stubarista (sta) that encodes the highly conserved putative ribosome-associated protein D-p40. sta maps to cytological position 2A3-B2 on the X chromosome and encodes a protein (D-p40) of 270 amino acids. D-p40 shares 63% identity with the human p40 ribosomal protein. P element-mediated transformation of a 4.4-kb genomic fragment encompassing the 1-kb transcript corresponding to D-p40 was used to rescue both a lethal (sta2) and a viable (sta1) mutation at the stubarista (sta) locus. Developmental analysis of the sta2 mutation implicates a requirement for D-p40 during oogenesis and imaginal development, which is consistent with the expression of sta throughout development. In addition, we have analyzed the basis of the sta1 visible phenotype which consists of shortened antennae and bristles. sta1 is a translocation of the 1E1-2 to 2B3-4 region of the X chromosome onto the third chromosome at 89B21-C4. We provide genetic evidence that Dp(1;3)sta1 is mutant at the spineless (ss) locus and that it is associated with partial D-p40 activity. We demonstrate that sta1 acts as a recessive enhancer of ss; reduction in the amount of D-p40 provided by the transposed X chromosomal region of sta1 reveals a haplo-insufficient phenotype of the otherwise recessive ss mutations. This phenomenon is reminiscent of the enhancing effect observed with Minute mutations, one of which, rp49, has previously been shown to encode a ribosomal protein.

Mutations in the protein phosphatase 1 gene at 87B can differentially affect suppression of position-effect variegation and mitosis in Drosophila melanogaster.

The suppressor of position effect variegation (PEV) locus Su-var(3)6 maps to 87B5-10. The breakpoints of deficiencies that define this interval have been placed on a 250-kb molecular map of the region. The locus is allelic to the ck19 complementation group previously shown to encode a type 1 serine-threonine protein phosphatase (PP1) catalytic subunit. When introduced into flies by P element-mediated transformation, a 5.8-kb genomic fragment carrying this gene overcomes the suppressor phenotype of Su-var(3)6(01) and recessive lethality of all mutations of the locus. Four of the mutant alleles at the locus show a broad correlation between high levels of suppression of PEV, a high frequency of aberrant mitosis and low PP1 activity in larval extracts. However, some alleles with low PP1 activity show weak suppression of PEV with a high frequency of abnormal mitosis, whereas others show strong suppression of PEV with normal mitosis. The basis for these discussed.

Analyses of PS integrin functions during Drosophila development.

The Drosophila position-specific (PS) antigens are homologues of the vertebrate integrins, a family of transmembrane proteins that function in cell-matrix and cell-cell adhesion. The common beta subunit of PS integrins (PS beta) is encoded by the lethal(l)myospheroid gene (mys) and is required during wing, eye and muscle development. By expressing PS beta protein at defined developmental periods, we have shown that PS integrins are required throughout pupation, but not earlier, for normal development of wings. In contrast, the key requirement for PS integrins in eye development occurs only in the late pupa. Furthermore, PS integrins are apparently not required for the differentiation of the ommatidial cells; only for their organization. These results are consistent with roles for PS integrins in the interactions between the wing epithelia during the two phases of pupal wing expansion and in maintaining the attachment of a fully formed fenestrated membrane to the basement membrane of the retina. We have also examined the functional significance of alternative splicing of the transcript of the mys gene using P element-mediated transformation to introduce transgenes producing only one of the two spliced forms of PS beta. We find that either form is sufficient to rescue postembryonic mys phenotypes in the wing, eye and muscle but that both of the two splice forms are necessary to rescue the mys embryonic defects. This result indicates a requirement for the alternative splicing of mys during embryogenesis. The location of the alternative exons suggests that the two forms of the PS beta integrin subunit may interact with alternative alpha subunits and/or ligands.

Cloning and molecular genetic analysis of Drosophila melanogaster interband DNA.

Interband DNA of Drosophila melanogaster polytene chromosomes was studied using a novel approach based on the electron microscopic (EM) analysis of chromosome regions carrying DNA fragments of known molecular genetic composition, inserted by P element-mediated transformation. Insertion of such fragments predominantly into interbands makes it possible to clone interband DNA by constructing genomic libraries from transformed strains and probing them with the insert DNA. The transformed strain P[H-sp70:Adh](61C) has insertion in the 61C7-8 interband on the left arm of chromosome 3. This DNA consists of part of the hsp70 gene promoter fused to the coding region of the Adh gene, and is flanked on either side by P element sequences. We constructed a genomic library from DNA of this strain and isolated a clone containing the insert and the interband DNA. Subsequently the genomic library of wild-type strain was probed with a subclone composed of interband DNA only. We have thus isolated a clone containing the entire native interband. 1289 bp of interband DNA was sequenced and found to be AT-rich (53.4%) with numerous regions of overlapping direct and inverted repeats, regulatory sites, and two overlapping open reading frames (ORFs).

Negative and Positive Regulators Modulate the Activity of a Silkmoth Chorion Gene during Choriogenesis

We have used in vitro linker substitution mutagenesis and P -element-mediated transformation in Drosophila to define cis -regulatory elements of the bidirectional promoter of the Bombyx mori A/B.L12 chorion genes in an in vivo situation. Within as essential 112 bp part of this promoter, mutations in two non-contiguous approximately 35 base-pair regions (A1 and A2) results in significant reduction of expression at late choriogenesis. The reduction occurs in both promoter orientations, indicating the existence of bidirectionally active positive elements. Mutations in A1, but not in A2, also lead to premature activation at early choriogenesis, suggesting the existence of a negative element essential for the finely tuned expression profile during choriogenesis. Normally, this element apparently prevents early expression; it also acts bidirectionally. The highly specific effects of these mutations suggest that several trans -acting regulators of chorion gene transcription have been conserved between silkmoths and fruitflies.