Maxizyme-mediated suppression of chikungunya virus replication and transmission in transgenic Aedes aegypti mosquitoes.

Chikungunya virus (CHIKV) is an emerging mosquito-borne pathogen of significant public health importance. There are currently no prophylactic vaccines or therapeutics available to control CHIKV. One approach to arbovirus control that has been proposed is the replacement of transmission-competent mosquitoes with those that are refractory to virus infection. Several transgene effectors are being examined as potentially useful for this population replacement approach. We previously demonstrated the successful use of hammerhead ribozymes (hRzs) as an antiviral effector transgene to control CHIKV infection of, and transmission by, Aedes mosquitoes. In this report we examine a maxizyme approach to enhance the catalytic activity and prevent virus mutants from escaping these ribozymes. We designed a maxizyme containing minimized (monomer) versions of two hRzs we previously demonstrated to be the most effective in CHIKV suppression. Three versions of CHIKV maxizyme were designed: Active (Mz), inactive (ΔMz), and a connected CHIKV maxizyme (cMz). The maxizymes with their expression units (Ae-tRNA val promoter and its termination signal) were incorporated into lentivirus vectors with selection and visualization markers. Following transformation, selection, and single-cell sorting of Vero cells, clonal cell populations were infected with CHIKV at 0.05 and 0.5 MOI, and virus suppression was assessed using TCID50-IFA, RT-qPCR, and caspase-3 assays. Five transgenic mosquito lines expressing cMz were generated and transgene insertion sites were confirmed by splinkerette PCR. Our results demonstrate that Vero cell clones expressing Mz exhibited complete inhibition of CHIKV replication compared to their respective inactive control version or the two parent hRzs. Upon oral challenge of transgenic mosquitoes with CHIKV, three out of the five lines were completely refractory to CHIKV infection, and all five lines tested negative for salivary transmission. Altogether, this study demonstrates that maxizymes can provide a higher catalytic activity and viral suppression than hRzs.

Transgenic and Diet-Enhanced Silk Production for Reinforced Biomaterials: A Metamaterial Perspective.

Silk fibers, which are protein-based biopolymers produced by spiders and silkworms, are fascinating biomaterials that have been extensively studied for numerous biomedical applications. Silk fibers often have remarkable physical and biological properties that typical synthetic materials do not exhibit. These attributes have prompted a wide variety of silk research, including genetic engineering, biotechnological synthesis, and bioinspired fiber spinning, to produce silk proteins on a large scale and to further enhance their properties. In this review, we describe the basic properties of spider silk and silkworm silk and the important production methods for silk proteins. We discuss recent advances in reinforced silk using silkworm transgenesis and functional additive diets with a focus on biomedical applications. We also explain that reinforced silk has an analogy with metamaterials such that user-designed atypical responses can be engineered beyond what naturally occurring materials offer. These insights into reinforced silk can guide better engineering of superior synthetic biomaterials and lead to discoveries of unexplored biological and medical applications of silk.

Antiviral Hammerhead Ribozymes Are Effective for Developing Transgenic Suppression of Chikungunya Virus in Aedes aegypti Mosquitoes.

The chikungunya virus (CHIKV) is an emerging pathogen with widespread distribution in regions of Africa, India, and Asia that threatens to spread into temperate climates with the introduction of its major vector, Aedes albopictus. CHIKV causes a disease frequently misdiagnosed as dengue fever, with potentially life-threatening symptoms that can result in a longer-term debilitating arthritis. The increasing risk of spread from endemic regions via human travel and commerce and the current absence of a vaccine put a significant proportion of the world population at risk for this disease. In this study we designed and tested hammerhead ribozymes (hRzs) targeting CHIKV structural protein genes of the RNA genome as potential antivirals both at the cellular and in vivo level. We employed the CHIKV strain 181/25, which exhibits similar infectivity rates in both Vero cell cultures and mosquitoes. Virus suppression assay performed on transformed Vero cell clones of all seven hRzs demonstrated that all are effective at inhibiting CHIKV in Vero cells, with hRz #9 and #14 being the most effective. piggyBac transformation vectors were constructed using the Ae. aegypti t-RNA(val) Pol III promoted hRz #9 and #14 effector genes to establish a total of nine unique transgenic Higgs White Eye (HWE) Ae. aegypti lines. Following confirmation of transgene expression by real-time polymerase chain reaction (RT-PCR), comparative TCID50-IFA analysis, in situ Immuno-fluorescent Assays (IFA) and analysis of salivary CHIKV titers demonstrated effective suppression of virus replication at 7 dpi in heterozygous females of each of these transgenic lines compared with control HWE mosquitoes. This report provides a proof that appropriately engineered hRzs are powerful antiviral effector genes suitable for population replacement strategies.

Suppression of the Arboviruses Dengue and Chikungunya Using a Dual-Acting Group-I Intron Coupled with Conditional Expression of the Bax C-Terminal Domain.

In portions of South Asia, vectors and patients co-infected with dengue (DENV) and chikungunya (CHIKV) are on the rise, with the potential for this occurrence in other regions of the world, for example the United States. Therefore, we engineered an antiviral approach that suppresses the replication of both arboviruses in mosquito cells using a single antiviral group I intron. We devised unique configurations of internal, external, and guide sequences that permit homologous recognition and splicing with conserved target sequences in the genomes of both viruses using a single trans-splicing Group I intron, and examined their effectiveness to suppress infections of DENV and CHIKV in mosquito cells when coupled with a proapoptotic 3' exon, ΔN Bax. RT-PCR demonstrated the utility of these introns in trans-splicing the ΔN Bax sequence downstream of either the DENV or CHIKV target site in transformed Aedes albopictus C6/36 cells, independent of the order in which the virus specific targeting sequences were inserted into the construct. This trans-splicing reaction forms DENV or CHIKV ΔN Bax RNA fusions that led to apoptotic cell death as evidenced by annexin V staining, caspase, and DNA fragmentation assays. TCID50-IFA analyses demonstrate effective suppression of DENV and CHIKV infections by our anti-arbovirus group I intron approach. This represents the first report of a dual-acting Group I intron, and demonstrates that we can target DENV and CHIKV RNAs in a sequence specific manner with a single, uniquely configured CHIKV/DENV dual targeting group I intron, leading to replication suppression of both arboviruses, and thus providing a promising single antiviral for the transgenic suppression of multiple arboviruses.

Targeted glycoengineering extends the protein N-glycosylation pathway in the silkworm silk gland.

The silkworm silk glands are powerful secretory organs that can produce and secrete proteins at high levels. As such, it has been suggested that the biosynthetic and secretory power of the silk gland can be harnessed to produce and secrete recombinant proteins in tight or loose association with silk fibers. However, the utility of the silkworm platform is constrained by the fact that it has a relatively primitive protein N-glycosylation pathway, which produces relatively simple insect-type, rather than mammalian-type N-glycans. In this study, we demonstrate for the first time that the silk gland protein N-glycosylation pathway can be glycoengineered. We accomplished this by using a dual piggyBac vector encoding two distinct mammalian glycosyltransferases under the transcriptional control of a posterior silk gland (PSG)-specific promoter. Both mammalian transgenes were expressed and each mammalian N-glycan processing activity was induced in transformed silkworm PSGs. In addition, the transgenic animals produced endogenous glycoproteins containing significant proportions of mammalian-type, terminally galactosylated N-glycans, while the parental animals produced none. This demonstration of the ability to glycoengineer the silkworm extends its potential utility as a recombinant protein production platform.

Disruption of dengue virus transmission by mosquitoes.

Current control efforts for mosquito-borne arboviruses focus on mosquito control involving insecticide applications, which are becoming increasingly ineffective and unsustainable in urban areas. Mosquito population replacement is an alternative arbovirus control concept aiming at replacing virus-competent vector populations with laboratory-engineered incompetent vectors. A prerequisite for this strategy is the design of robust anti-pathogen effectors that can ultimately be genetically driven through a wild-type population. Several anti-pathogen effector concepts have been developed that target the RNA genomes of arboviruses such as dengue virus in a highly sequence-specific manner. Design principles are based on long inverted-repeat RNA triggered RNA interference, catalytic hammerhead ribozymes, and trans-splicing Group I Introns that are able to induce apoptosis in virus-infected cells following splicing with target viral RNA.

Trans-splicing group I intron targeting hepatitis C virus IRES mediates cell death upon viral infection in Huh7.5 cells.

The HCV-IRES sequence is vital for both protein translation and genome replication and serves as a potential target for anti-HCV therapy. We constructed a series of anti-HCV group I introns (αHCV-GrpIs) to attack conserved target sites within the HCV IRES. These αHCV-GrpIs were designed to mediate a trans-splicing reaction that replaces the viral RNA genome downstream of the 5' splice site with a 3' exon that encodes an apoptosis-inducing gene. Pro-active forms of the apoptosis inducing genes BID, Caspase 3, Caspase 8, or tBax were modified by incorporation of the HCV NS5A/5B cleavage sequence in place of their respective endogenous cleavage sites to ensure that only HCV infected cells would undergo apoptosis following splicing and expression. Huh7.5 cells transfected with each intron were challenged at MOI 0.1 with HCV-Jc1FLAG2 which expresses a Gaussia Luciferase (GLuc) marker. Virus-containing supernatants were then assayed for GLuc expression as a measure of viral replication inhibition. Cellular extracts were analyzed for the presence of correct splice products by RT-PCR and DNA sequencing. We also measured levels of Caspase 3 activity as a means of quantifying apoptotic cell death. Each of these αHCV-GrpI introns was able to correctly splice their 3' apoptotic exons onto the virus RNA genome at the targeted Uracil, and resulted in greater than 80% suppression of the GLuc marker. A more pronounced suppression effect was observed with TCID50 virus titrations, which demonstrated that these αHCV-GrpIs were able to suppress viral replication by more than 2 logs, or greater than 99%. Robust activation of the apoptotic factor within the challenged cells was evidenced by a significant increase of Caspase 3 activity upon viral infection compared to non-challenged cells. This novel genetic intervention tool may prove beneficial in certain HCV subjects.

Effective suppression of dengue virus using a novel group-I intron that induces apoptotic cell death upon infection through conditional expression of the Bax C-terminal domain.

INTRODUCTION: Approximately 100 million confirmed infections and 20,000 deaths are caused by Dengue virus (DENV) outbreaks annually. Global warming and rapid dispersal have resulted in DENV epidemics in formally non-endemic regions. Currently no consistently effective preventive measures for DENV exist, prompting development of transgenic and paratransgenic vector control approaches. Production of transgenic mosquitoes refractory for virus infection and/or transmission is contingent upon defining antiviral genes that have low probability for allowing escape mutations, and are equally effective against multiple serotypes. Previously we demonstrated the effectiveness of an anti-viral group I intron targeting U143 of the DENV genome in mediating trans-splicing and expression of a marker gene with the capsid coding domain. In this report we examine the effectiveness of coupling expression of ΔN Bax to trans-splicing U143 intron activity as a means of suppressing DENV infection of mosquito cells. RESULTS: Targeting the conserved DENV circularization sequence (CS) by U143 intron trans-splicing activity appends a 3' exon RNA encoding ΔN Bax to the capsid coding region of the genomic RNA, resulting in a chimeric protein that induces premature cell death upon infection. TCID50-IFA analyses demonstrate an enhancement of DENV suppression for all DENV serotypes tested over the identical group I intron coupled with the non-apoptotic inducing firefly luciferase as the 3' exon. These cumulative results confirm the increased effectiveness of this αDENV-U143-ΔN Bax group I intron as a sequence specific antiviral that should be useful for suppression of DENV in transgenic mosquitoes. Annexin V staining, caspase 3 assays, and DNA ladder observations confirm DCA-ΔN Bax fusion protein expression induces apoptotic cell death. CONCLUSION: This report confirms the relative effectiveness of an anti-DENV group I intron coupled to an apoptosis-inducing ΔN Bax 3' exon that trans-splices conserved sequences of the 5' CS region of all DENV serotypes and induces apoptotic cell death upon infection. Our results confirm coupling the targeted ribozyme capabilities of the group I intron with the generation of an apoptosis-inducing transcript increases the effectiveness of infection suppression, improving the prospects of this unique approach as a means of inducing transgenic refractoriness in mosquitoes for all serotypes of this important disease.

Design and analysis of hammerhead ribozyme activity against an artificial gene target.

In vitro cleavage assays are routinely conducted to properly assess the catalytic activity of hammerhead ribozymes (HHR) against target RNA molecules like dengue virus RNA. These experiments are performed for initial assessment of HHR catalysis in a cell-free system and have been simplified by the substitution of agarose gel electrophoresis for SDS-PAGE. Substituting mobility assays enables the analysis of ribozymes in a more rapid fashion without radioisotopes. Here we describe the in vitro transcription of an HHR and corresponding target from T7-promoted plasmids into RNA molecules leading to the analysis of HHR activity against the RNA target by in vitro cleavage assays.

A novel dengue virus detection method that couples DNAzyme and gold nanoparticle approaches.

BACKGROUND: Recent epidemics of dengue viruses (DENV) coupled with new outbreaks on the horizon have renewed the demand for novel detection methods that have the ability to identify this viral pathogen prior to the manifestation of symptoms. The ability to detect DENV in a timely manner is essential for rapid recovery from disease symptoms. A modified lab-derived 10-23 DNAzyme tethered to gold nanoparticles provides a powerful tool for the detection of viruses, such as DENV. RESULTS: We examined the effectiveness of coupling DNAzyme (DDZ) activation to the salt-induced aggregation of gold nanoparticles (AuNP) to detect dengue virus (DENV) progeny in mosquito cells. A DNAzyme was designed to recognize the 5' cyclization sequence (5' CS) that is conserved among all DENV, and conjugated to AuNPs. DDZ-AuNP has demonstrated the ability to detect the genomic RNA of our model dengue strain, DENV-2 NGC, isolated from infected Aedes albopictus C6/36 cells. These targeting events lead to the rapid aggregation of AuNPs, resulting in a red to clear color transition of the reaction mixes, and thus positive detection of the DENV RNA genome. The inclusion of SDS in the reaction mixture permitted the detection of DENV directly from cell culture supernatants without additional sample processing. Specificity assays demonstrated detection is DENV-specific, while sensitivity assays confirm detection at levels of 1 × 10(1) TCID50 units. These results demonstrate DDZ-AuNP effectively detects DENV genomes in a sequence specific manner and at concentrations that are practical for field use. CONCLUSIONS: We have developed an effective detection assay using DNAzyme catalysis coupled with AuNP aggregation for the detection of DENV genomes in a sequence specific manner. Full development of our novel DDZ-AuNP detection method will provide a practical, rapid, and low cost alternative for the detection of DENV in mosquito cells and tissues, and possibly infected patient serum, in a matter of minutes with little to no specialized training required.

Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties.

The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors to create transgenic silkworms encoding chimeric silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric silkworm/spider silk proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental silkworm silk fibers and as tough as native dragline spider silk fibers. These results demonstrate that silkworms can be engineered to manufacture composite silk fibers containing stably integrated spider silk protein sequences, which significantly improve the overall mechanical properties of the parental silkworm silk fibers.

Insect transgenesis: current applications and future prospects.

The ability to manipulate the genomes of many insects has become a practical reality over the past 15 years. This has been led by the identification of several useful transposon vector systems that have allowed the identification and development of generalized, species-specific, and tissue-specific promoter systems for controlled expression of gene products upon introduction into insect genomes. Armed with these capabilities, researchers have made significant strides in both fundamental and applied transgenics in key model systems such as Bombyx mori, Tribolium casteneum, Aedes aegypti, and Anopheles stephensi. Limitations of transposon systems were identified, and alternative tools were developed, thus significantly increasing the potential for applied transgenics for control of both agricultural and medical insect pests. The next 10 years promise to be an exciting time of transitioning from the laboratory to the field, from basic research to applied control, during which the full potential of gene manipulation in insect systems will ultimately be realized.

Targeting of highly conserved Dengue virus sequences with anti-Dengue virus trans-splicing group I introns.

BACKGROUND: Dengue viruses (DENV) are one of the most important viral diseases in the world with approximately 100 million infections and 200,000 deaths each year. The current lack of an approved tetravalent vaccine and ineffective insecticide control measures warrant a search for alternatives to effectively combat DENV. The trans-splicing variant of the Tetrahymena thermophila group I intron catalytic RNA, or ribozyme, is a powerful tool for post-transcriptional RNA modification. The nature of the ribozyme and the predictability with which it can be directed makes it a powerful tool for modifying RNA in nearly any cell type without the need for genome-altering gene therapy techniques or dependence on native cofactors. RESULTS: Several anti-DENV Group I trans-splicing introns (αDENV-GrpIs) were designed and tested for their ability to target DENV-2 NGC genomes in situ. We have successfully targeted two different uracil bases on the positive sense genomic strand within the highly conserved 5'-3' cyclization sequence (CS) region common to all serotypes of DENV with our αDENV-GrpIs. Our ribozymes have demonstrated ability to specifically trans-splice a new RNA sequence downstream of the targeted site in vitro and in transfected insect cells as analyzed by firefly luciferase and RT-PCR assays. The effectiveness of these αDENV-GrpIs to target infecting DENV genomes is also validated in transfected or transformed Aedes mosquito cell lines upon infection with unattenuated DENV-2 NGC. CONCLUSIONS: Analysis shows that our αDENV-GrpIs have the ability to effectively trans-splice the DENV genome in situ. Notably, these results show that the αDENV-GrpI 9v1, designed to be active against all forms of Dengue virus, effectively targeted the DENV-2 NGC genome in a sequence specific manner. These novel αDENV-GrpI introns provide a striking alternative to other RNA based approaches for the transgenic suppression of DENV in transformed mosquito cells and tissues.

Use of the piggyBac transposon to create HIV-1 gag transgenic insect cell lines for continuous VLP production.

BACKGROUND: Insect baculovirus-produced Human immunodeficiency virus type 1 (HIV-1) Gag virus-like-particles (VLPs) stimulate good humoral and cell-mediated immune responses in animals and are thought to be suitable as a vaccine candidate. Drawbacks to this production system include contamination of VLP preparations with baculovirus and the necessity for routine maintenance of infectious baculovirus stock. We used piggyBac transposition as a novel method to create transgenic insect cell lines for continuous VLP production as an alternative to the baculovirus system. RESULTS: Transgenic cell lines maintained stable gag transgene integration and expression up to 100 cell passages, and although the level of VLPs produced was low compared to baculovirus-produced VLPs, they appeared similar in size and morphology to baculovirus-expressed VLPs. In a murine immunogenicity study, whereas baculovirus-produced VLPs elicited good CD4 immune responses in mice when used to boost a prime with a DNA vaccine, no boost response was elicited by transgenically produced VLPs. CONCLUSION: Transgenic insect cells are stable and can produce HIV Pr55 Gag VLPs for over 100 passages: this novel result may simplify strategies aimed at making protein subunit vaccines for HIV. Immunogenicity of the Gag VLPs in mice was less than that of baculovirus-produced VLPs, which may be due to lack of baculovirus glycoprotein incorporation in the transgenic cell VLPs. Improved yield and immunogenicity of transgenic cell-produced VLPs may be achieved with the addition of further genetic elements into the piggyBac integron.

Effective suppression of Dengue fever virus in mosquito cell cultures using retroviral transduction of hammerhead ribozymes targeting the viral genome.

Outbreaks of Dengue impose a heavy economic burden on developing countries in terms of vector control and human morbidity. Effective vaccines against all four serotypes of Dengue are in development, but population replacement with transgenic vectors unable to transmit the virus might ultimately prove to be an effective approach to disease suppression, or even eradication. A key element of the refractory transgenic vector approach is the development of transgenes that effectively prohibit viral transmission. In this report we test the effectiveness of several hammerhead ribozymes for suppressing DENV in lentivirus-transduced mosquito cells in an attempt to mimic the transgenic use of these effector molecules in mosquitoes. A lentivirus vector that expresses these ribozymes as a fusion RNA molecule using an Ae. aegypti tRNA(val) promoter and terminating with a 60A tail insures optimal expression, localization, and activity of the hammerhead ribozyme against the DENV genome. Among the 14 hammerhead ribozymes we designed to attack the DENV-2 NGC genome, several appear to be relatively effective in reducing virus production from transduced cells by as much as 2 logs. Among the sequences targeted are 10 that are conserved among all DENV serotype 2 strains. Our results confirm that hammerhead ribozymes can be effective in suppressing DENV in a transgenic approach, and provide an alternative or supplementary approach to proposed siRNA strategies for DENV suppression in transgenic mosquitoes.

piggyBac is an effective tool for functional analysis of the Plasmodium falciparum genome.

BACKGROUND: Much of the Plasmodium falciparum genome encodes hypothetical proteins with limited homology to other organisms. A lack of robust tools for genetic manipulation of the parasite limits functional analysis of these hypothetical proteins and other aspects of the Plasmodium genome. Transposon mutagenesis has been used widely to identify gene functions in many organisms and would be extremely valuable for functional analysis of the Plasmodium genome. RESULTS: In this study, we investigated the lepidopteran transposon, piggyBac, as a molecular genetic tool for functional characterization of the Plasmodium falciparum genome. Through multiple transfections, we generated 177 unique P. falciparum mutant clones with mostly single piggyBac insertions in their genomes. Analysis of piggyBac insertion sites revealed random insertions into the P. falciparum genome, in regards to gene expression in parasite life cycle stages and functional categories. We further explored the possibility of forward genetic studies in P. falciparum with a phenotypic screen for attenuated growth, which identified several parasite genes and pathways critical for intra-erythrocytic development. CONCLUSION: Our results clearly demonstrate that piggyBac is a novel, indispensable tool for forward functional genomics in P. falciparum that will help better understand parasite biology and accelerate drug and vaccine development.

Intramolecular integration assay validates integrase phi C31 and R4 potential in a variety of insect cells.

Phage phi C31 and R4 integrases are site-specific and unidirectional serine recombinases. We have analyzed the ability of these integrases to mediate intramolecular integration between their attB and attP sites in 7 important insect cell lines as a means of predicting their relative mobility in the corresponding insect species. Both integrases exhibit significantly higher frequencies in Drosophila S2 cells than in the other insect cell lines examined, but do work well in all of the species tested. Our results, coupled with previous results of the activity of phi C31 integrase in D. melanogaster and Aedes aegypti, suggest the family of serine catalyzed integrases will be useful site-specific integration tools for functional genome analysis and genetic engineering in a wide range of insect species.

Examining the relative activity of several dicistrovirus intergenic internal ribosome entry site elements in uninfected insect and mammalian cell lines.

Comparisons of the relative activities of 11 intergenic region (IGR) internal ribosome entry site (IRES) elements of insect dicistrovirus with 5' IRES elements of the hepatitis C and encephalomyocarditis viruses were performed in insect and mammalian cells. Dual luciferase assays were performed to determine the most effective dicistrovirus IGR IRES in the lepidopteran cell lines Sf9 (Spodoptera frugiperda) and BmN (Bombyx mori), and the dipteran cell lines S2 (Drosophila melanogaster) and ATC-10 (Aedes aegypti). Evaluation of dual luciferase expression from DNA plasmids and in vitro-transcribed RNA revealed apparent splicing with certain IRES elements. Though IRES activity depended upon the cell line examined, the black queen cell and Drosophila C dicistrovirus intergenic IRES elements were most effective for coupled gene expression in the diverse insect cell lines examined.

Analysis of the piggyBac transposase reveals a functional nuclear targeting signal in the 94 c-terminal residues.

BACKGROUND: The piggyBac transposable element is a popular tool for germ-line transgenesis of eukaryotes. Despite this, little is known about the mechanism of transposition or the transposase (TPase) itself. A thorough understanding of just how piggyBac works may lead to more effective use of this important mobile element. A PSORTII analysis of the TPase amino acid sequence predicts a bipartite nuclear localization signal (NLS) near the c-terminus, just upstream of a putative ZnF (ZnF). RESULTS: We fused the piggyBac TPase upstream of and in-frame with the enhanced yellow fluorescent protein (EYFP) in the Drosophila melanogaster inducible metallothionein protein. Using Drosophila Schneider 2 (S2) cells and the deep red fluorescent nuclear stain Draq5, we were able to track the pattern of piggyBac localization with a scanning confocal microscope 48 hours after induction with copper sulphate. CONCLUSION: Through n and c-terminal truncations, targeted internal deletions, and specific amino acid mutations of the piggyBac TPase open reading frame, we found that not only is the PSORTII-predicted NLS required for the TPase to enter the nucleus of S2 cells, but there are additional requirements for negatively charged amino acids a short length upstream of this region for nuclear localization.

Mutational analysis of highly conserved aspartate residues essential to the catalytic core of the piggyBac transposase.

BACKGROUND: The piggyBac mobile element is quickly gaining popularity as a tool for the transgenesis of many eukaryotic organisms. By studying the transposase which catalyzes the movement of piggyBac, we may be able to modify this vector system to make it a more effective transgenesis tool. In a previous publication, Sarkar A, Sim C, Hong YS, Hogan JR, Fraser MJ, Robertson HM, and Collins FH have proposed the presence of the widespread 'DDE/DDD' motif for piggyBac at amino acid positions D268, D346, and D447. RESULTS: This study utilizes directed mutagenesis and plasmid-based mobility assays to assess the importance of these residues as the catalytic core of the piggyBac transposase. We have functionally analyzed individual point-mutations with respect to charge and physical size in all three proposed residues of the 'DDD' motif as well as another nearby, highly conserved aspartate at D450. All of our mutations had a significant effect on excision frequency in S2 cell cultures. We have also aligned the piggyBac transposase to other close family members, both functional and non-functional, in an attempt to identify the most highly conserved regions and position a number of interesting features. CONCLUSION: We found all the designated DDD aspartates reside in clusters of amino acids that conserved among piggyBac family transposase members. Our results indicate that all four aspartates are necessary, to one degree or another, for excision to occur in a cellular environment, but D450 seems to have a tolerance for a glutamate substitution. All mutants tested significantly decreased excision frequency in cell cultures when compared with the wild-type transposase.