Progress 10/01/07 to 09/30/12
Outputs
OUTPUTS: During the course of the entire project I undertook experiments aimed at identifying and isolating new transposons that could be used in agricultural and biomedical research and practice and also determined how some of these function and how they interact with the host genomes. During the project period I have given numerous invited presentations to national and international professional societies, academic departments through invited departmental seminars, and been an ongoing member of the Research Coordination Meeting for improving the Sterile Insect Technique for the IAEA. I have also served on and chaired NIH study sections. I also supervised the Culex quinquefasciatus genome project from 2005-2010 which provided an important community resource for vector biologists. PARTICIPANTS: Other UCR Collaborators: Susan R. Wessler (Botany and Plant Sciences), Jason E. Stajich (Plant Pathology and Microbiology), Marylynn Yates (Environmental Sciences), Anil Deolaliker (Economics), Mary Gauvain (Psychology), Sharon Walker (Engineering), Mark Matsumoto (Engineering), Ashok Mulchandani (Engineering). Non-UCR collaborators: Nancy L. Craig (Johns Hopkins School of Medicine/HHMI), Patricia Conrad (UC Davis Veterinary Medicine), Michael Wilkes (UC Davis School of Medicine), Gerald Franz, IAEA, Austria. Technical Staff: Robert H. Hice, SRA V. TARGET AUDIENCES: The target audiences for the past five years were members of professional communities, scientists, faculty, students and members of IAEA panels. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The outcomes of my work during this time were the discovery of new transposable elements from insects that are mobile in vertebrate and invertebrate hosts with the TcBuster transposon being highly active in human cells and so a candidate for gene therapy research. Another outcome of my work has been the analysis of the piRNA complement of mosquitoes in order to design transposons that can evade this immune response system and this now forms the basis of a new CRIS project. Anther outcome was the publication of the Culex genome in 2010; the 3rd mosquito genome sequenced. The database is now publicly searchable through public access.
Publications
- Woodward, L. E., X. Li, N. Malani, A. Kaja, R. H. Hice, P. W. Atkinson, F. D. Bushman, N. L. Craig, M. H. Wilson, (2012). Comparative analysis of the recently discovered hAT transposon TcBuster in human cells. PLoS One 7:e42666. PMD: 23166581. Li, X., H. Ewis, R. H. Hice, N. Malani, N. Parker, L. Zhou, C. Feshotte, F. D. Bushman, P. W. Atkinson, N. L. Craig (2013). A resurrected mammalian hAT transposable element and a closely related insect element are highly active in human cell culture. Proc Natl. Acad. Sci USA 110: E487-487. PMD: 23091042. Arensburger, P., R. H. Hice, J. A. Wright, N. L. Craig, P. W. Atkinson (2011). The mosquito Aedes aegypti has a large genome size and high transposable element load but contains a low proportion of transposon-specific piRNAs. BMC Genomics 12: doi.1186/1471-2164-12-606. PMD: 22171608. Kim, Y.-J., R. H. Hice, D. A. OBrochta, P. W. Atkinson (2011). DNA sequence requirements for hobo transposable element transposition in Drosophila. Genetica 139: 985-997. Khalon, A. S., R. H. Hice, D. A. OBrochta, P. W. Atkinson (2011). DNA binding activities of the Herves transposase from the mosquito Anopheles gambiae. Mobile DNA 2(1):9. Arensburger, P, R. H. Hice, L. Zhou, R. C. Smith, A. C. Tom, J. A. Wright, J. Knapp, D. A. OBrochta, N. L. Craig, P. W. Atkinson (2011). Phylogenetic and functional characterization of the hAT transposon superfamily. Genetics 188: 45-57. Arensburger P.et al., (2010). Sequencing of Culex quinquefasciatus establishes a platform for mosquito comparative genomics. Science 330. 86-88. Bartholomay, L.C,. et al. (2010). Pathogenomics of Culex quinquefasciatus and meta-analysis of infection responses to diverse pathogens. Science 330. 88-90 Smith, R. C, P. W. Atkinson (2010). Mobility properties of the Hermes transposable element in transgenic lines of Aedes aegypti. Genetica July 3. OBrochta, D. A., C. D. Stosic, K. Pillit, R. A. Subramanian, R. H. Hice, P. W. Atkinson (2009). Transpositionally active episomal hAT elements. BMC Mol. Biol. 10: 108. Subramanian, R. A., L. A. Cathcart, E. S. Krafsur, P. W. Atkinson, D. A. OBrochta (2009). Hermes transposon distribution in Musca domestica. J. Hered. 100: 473-480 Atkinson, P. W. Proposed uses of transposons in insect and medical biotechnology (2008). Adv. Exp. Med. Biol. 627: 60-70. Subramanian, R. A., P. Arensburger, P. W. Atkinson and D. A. OBrochta. (2007). Transposable element dynamics of the hAT element Herves in the human malaria vector Anopheles gambiae s.s. Genetics. 176:2477-2487. Nene, V., J. R. Wortman, D. Lawson, B. Haas, C. Kodira, Z. Tu, B. Loftus, Z. Xi, K. Megy, M. Grabherr, Q. Ren, E. M. Zdobnov, N. F. Lobo, K. S. Campbell, S. E. Brown, M. F. Bonaldo, J. Zhu, S. P. Sinkins et al., (2007). Genome sequence of Aedes aegypti, a major arbovirus vector. Science 316:1718-1723. Smith, R. C., M. F. Walter, R. H. Hice, D. A. OBrochta and P. W. Atkinson (2007). Testes-specific expression of the beta2 tubulin promoter of Aedes aegypti and its application as a genetic sex-separation marker. Insect Mol. Biol. 16: 61-71. OBrochta, D. A., Subramanian, R. A., Orsetti, J., Peckham, E., Nolan, N., Arensburger, P., Atkinson, P. W. and J. D. Charlwood. (2006). hAT element population genetics in Anopheles gambiae s.l. in Mozambique. Genetica 127:185-198. Wang, X. H., R. Aliyari, . X. Li, H. W. Li, K. Kim, R. Carthew, P. Atkinson, and S. W. Ding (2006). RNA interference directs innate immunity in viruses in adult Drosophila. Science 312:452-454.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: The outputs of the entire project have been reported to local, national and international meetings of learned societies and published in peer-reviewed journals. One patent was applied for. The small RNA research has been particularly well received and was selected as a talk at an international Keystone symposium. The research has also been presented in grant submissions to NIH. I also presented an overview of genomics research and vector biology research underway at UCR to scientists and research leaders in Tanzania, Kenya and India as part of a UCR-UCD delegation. PARTICIPANTS: Robert Hice, SRA UCR Jennifer Wright, GSR, UCR Joshua Knapp, GSR, UCR Amandeep Kahlon, GSR, UCR. Peter Arensburger, PD, UCR, David OBrochta, Professor, U Maryland Nancy Craig, Professor, JHMI/HHMI TARGET AUDIENCES: Learned scientific societies, scientists, researchers and students. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
My research has continued to focus on developing transposon-based genetic tools for insects of agricultural and medical importance. To this end I have continued to discover new transposon active in insects and also in other systems as well as determining how they function both in vivo and in vitro. I have also commenced characterization of the piRNA system of insects in order to determine how this small RNA system regulates transposons within a genome with the aim being to develop transposons that can overcome it. The result will be highly active transposons that can function in target species and will lead to new strategies for genetic control of insects.
Publications
- 1. Arensburger, P., R. H. Hice, J. A. Wright, N. L. Carig and P. W. Atkinson. (2011). The mosquito Aedes aegypti has a large genome size and high transposable element load but contains a low proportion of transposon-specific piRNAs. BMC Genomics. 12:606. 2. Kim, Y.-J., R. H. Hice, D. A. OBrochta and P. W. Atkinson (2011). DNA sequence requirements for hobo element transposition in Drosophila melanogaster. Genetica 139: 985-997. 3. Kahlon, A. S., R. H. Hice, D. A. OBrochta and P. W. Atkinson (2011). DNA binding activities of the Herves transposase from the mosquito Anopheles gambiae. Mob DNA 2:9. 4. Arensburger, P., R. H. Hice, L. Zhou, R. C. Smith, A. C. Tom, J. A. Wright, J. Knapp, D. A. OBrochta, N. L. Craig and P. W. Atkinson. (2011). Phylogenetic and functional characterization of the hAT transposon superfamily. Genetics. 188: 45-57.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: My research outputs for the 2010 year were reported at the second American Society for Microbiology meeting in Montreal in April, through an internal UCR seminar to the Center for Plant Cell Biology in November and through publications in peer reviewed scientific journals. In addition, as Director of the Center for Disease Vector Research at UCR, I have promoted the Center and the campus by participating in publications and videos (on YouTube). In addition I am involved in the UC Global Health Institute as a member of the steering committee and this covers, amongst other issues, health through agriculture. The UCGHI disseminates information through brochures and their web site. While diverse, all of these are associated with more core research of developing genetic tools in insects for use in agriculture and medicine. I was also the sole editor of a book entitled "Vector Biology, Ecology and Control". PARTICIPANTS: There were a large number of authors on the genome projects from a large number of institutions both within the US and from Europe and I will not list them here. My long term collaborators are Dr. David A. O'Brochta from the Department of Entomology, University of Maryland and Dr. Nancy L. Craig from the Department of Molecular Biology and Genetics and HHMI at Johns Hopkins School of Medicine. TARGET AUDIENCES: My target audience is the scientific community, undergraduate and graduate students at UCR and, through the UCGHI, other UC campuses. My clientele are citizens and primary producers whose livelihood and/or health are disaffected by insect pests either directly or indirectly through the use of chemical control agents. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The main outcome was the completion and publication of the genome project for Culex quinquefasciatus, a project which had been underway for some five years. The description of this genome provides researchers with the foundation from which genes and pathways can be identified in this mosquito pest and new approaches to its control undertaken. The second paper in this series describes the characterization of genes involved in the pathobiology of Culex. The published book describes a range of approaches and outcomes in vector biology centered around a broad definition of biological control.
Publications
- Arensburger P.et al., (2010). Sequencing of Culex quinquefasciatus establishes a platform for mosquito comparative genomics. Science 330. 86-88. Bartholomay, L.C,. et al. (2010). Pathogenomics of Culex quinquefasciatus and meta-analysis of infection responses to diverse pathogens. Science 330. 88-90 Smith, R. C, P. W. Atkinson (2010). Mobility properties of the Hermes transposable element in transgenic lines of Aedes aegypti. Genetica July 3. Atkinson, P. W. (ed.) (2010). Vector Biology and Control. Springer, 260 pp.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: In addition to publications (below) I presented data at: 1. An invited seminar to the Biology Dept at Kansas State, February, 2. An invited seminar to the CDVR, UC Riverside, April. 3. An invited seminar to the Plant Pathology and Microbiology Dept, UCR, October. 4. An invited seminar to a Next Gen sequencing workshop in the IIGB, UCR, November. One of the transposons discovered and partially characterized here (called TcB) is being tested by a company (Transposagen) with discussion of a patent being filed. PARTICIPANTS: Peter Atkinson, Principal Investigator. Peter Arensburger, Research Specialist. Robert Hice, SRA. Stephanie Russell, SRA. Jennifer Wright, GSR (Entomology). Joshua Knapp, GSR (Biochemistry and Molecular Biology). Amandeep Kahlon, GSR (Cell, Molecular and Developmental Biology). Nancy L. Craig, Professor, HHMI/Johns Hopkins School of Medicine (Co-PI and collaborator). David A. O'Brochta, University of Maryland Biotechnology Institute (Co-PI and collaborator). TARGET AUDIENCES: My target audience is the scientific community and reporting to it was covered on the first page. My research produces genetic tools and knowledge that will be of use to the scientific community and, ultimately, industry and medicine. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
In the past year we have achieved progress on a number of fronts. The Culex pipiens quinquefascistus genome project has been completed with manuscripts in preparation and near submission. This Culex genome resource is now widely used by the insect community through the relevant genome pages maintained at NCBI, EBI and VectorBase. We embarked on the analysis of small RNA sequences from the mosquito Aedes aegypti in an effort to characterize the piRNA system of this medically important mosquito and have shown that this transposon-rich insect has a very high piRNA load, the majority of which are targeted to the transposons of the mosquito. We also demonstrated that piRNAs are generated to transgenic transposases we had previously inserted into this mosquito. We are now determining whether these piRNAs can explain, at least in part, the silencing of transgenic transposons in this species. Our work on the study of the structure and function of active transposons has continued on the Buster and Ac members of the hAT transposon superfamily. Together with our collaborator Nancy L. Craig of HHMI/Johns Hopkins we have identified and characterized hyperactive forms of these transposase using yeast-based genetic screens and we are now testing these in mosquitoes and Drosophila. We have also identified a mutant with an altered target site insertion sequence which may enable us to to locate, at least in part, the target DNA binding domain of the transposase. Modification of this may result in the development of transposases with increased target site specificity which would then by used as more precise tools in genetic manipulations. We have also determined the transposase binding sites of the Herves transposon which is an active transposon from the malaria vector Anopheles gambiae.
Publications
- Atkinson PW (ed.) (2010). Vector Biology, Ecology and Control. Springer Publishers. 260pp.
- Smith RC, Atkinson PW (2010) Mobility properties of the Hermes transposable element in transgenic lines of Aedes aegypti. Genetica.
- O'Brochta DA. Stosic, CD, Pilitt, K, Subramanian RA, Hice RH, Atkinson PW (2010). Transpositionally active episomal hAT elements. BMC Mole. Biol.
- Subramanian RA, Cathcart LA, Krasfur ES, Atkinson PW, O'Brochta DA (2009). Hermes transposon distribution and structure in Musca domestica. J. Hered. 100:473-480.
- Cruz J., Sieglaff DH, Arensburger P, Atkinson PW, Raikhel AS (2009). Nuclear receptors in the mosquito Aedes aegypti: annotation, hormonal regulation and expression profiling. FEBS J. 276: 1233-54.
- Atkinson PW, O'Brochta DA (2009). Genetic Engineering. In "Encyclopedia of Insects" 2nd ed. Eds: V Resh and R. Carde (article reprinted from 1st ed.). Academic Press.
- Arensburger P, Atkinson PW (2009). Genomics. In "Encyclopedia of Insects" 2nd ed. Eds: V Resh and R. Carde. Academic Press.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: In the past 12 months the following outputs have been completed. Activities. I mentored three graduate students and taught in Molecular Entomology, Molecular biology and Advanced Molecular Biology. I was asked to chair a review of the USDA Program in Fargo, ND but declined due to my role as Chair of the NIH Study section in Vector Biology. I also declined invited service on the USDA Genomics panel for this same reason. I continued to conduct experiments on the molecular basis of transposition and its application in insect control. Events. In June 2008 I co-organized an international meeting - International workshop on Invertebrate Transgenesis and Genomic - which was held at Asilomar, California. The meeting was partially supported by the USDA and attracted approximately sixty researchers. I attended a Mobile DNA conferences in France in April and presented data and did likewise at a workshop in Woods Hole in October. Services. None outside of normal UCR internal activities. Products. A non-disclosure document for the use of a new active transposon, TcBuster, was submitted to UC and NIH. Dissemination. None outside of my usual academic presentations. PARTICIPANTS: Individuals. Peter Atkinson, PI Robert Hice, SRA Stephanie Russell, SRA Amandeep Kahlon, GSR Joshua Knapp, GSR Jennifer Wright, GSR Collaborators. David O'Brochta Nancy Craig. Partner Organizations. University of Maryland Johns Hopkins School of Medicine TARGET AUDIENCES: Students of the University of California (lab and classroom instruction). Research scientists and students in the same and related areas (research presentations). PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
In 2008 we demonstrated that the TcBuster transposon is active in human cells at a frequency that makes it attractive for use in human gene therapy. This transposon is also active in insect cells. We generated, identified and isolated hyperactive mutants of the Hermes transposon and are now testing these for activity in flies and mosquitoes. We determined the DNA binding properties of the Herves transposase in mosquitoes and, using new mutants of Hermes, developed a model for the binding of this transposase to its target DNA. We generated piRNA molecules from both wild type and transgenic mosquitoes with the aim being to determine if these small RNAs regulate transposon activity in these insects. Addressing this issue successfully may enable the use of transposons as genetic tools in mosquitoes and perhaps other insects in which their activity is silenced. Preliminary results show that small RNAs are generated to transposons as well as to recently introduced foreign transposons.
Publications
- Ray, D. A., C. Feschotte, H. J. Pagan, J. D. Smith, E. Pritham, P. Arensburger, P. W. Atkinson and N. L. Craig. (2008). Multiple waves of recent DNA transposon activity in the bat, Myotis lucifugus. Genome Res. 18:717-728.
- Atkinson, P. W. (2008). Proposed uses of transposons in insect and medical biotechnology. Adv. Exp. Med. Biol. 627: 60-70.
- Lawson, D., P. Arensburger, P. Atkinson, N. J. Besansky, R. V. Bruggner, R. Butler, K. S. Campbell, G. K. Christopides, S. Christley, E. Dialynas, M. Hammnod, C. A. Hill, N. Konopinski, N. F. Lobo, R. M. MacCallum, G. Madey, K. Megy, J. Meyer, S. Redmond, D. W. Severson, E. O. Stinson, P. Topalis, E. Birney, W. M. Gelbart, F. C. Kafatos, C. Louis, and F. H. Collins. (2008). VectorBase: a data resource for invertebrate genomics. Nucl. Acids Res. (in press).
- Triboilum Genome Sequencing Consortium (2008). The genome of the model beetle and pest Tribolium castaneum. Nature, 452:949-055.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: In the past twelve months we have published five papers and presented our research at the FASEB meeting on human mobile DNA held at Tuscon, AZ in June of 2007. Two students obtained their PhDs and presented their findings in seminars at UCR. An overview of the genomics field as applied to entomology was presented at the annual ESA meeting in San Diego in December and to the MVCAC in Palm Springs in November. We also oversee the Culex pipiens quinquefasciatus genome project and this is now publicly available at NCBI.
PARTICIPANTS: Robert Hice, Stephanie Russell, Peter Atkinson, Ryan Smith, Ala Perumalsammy, Lisa Friedli, Joshua Knapp, Peter Arensburger, Amandeep Kahlon (all UCR); David OBrochta (University of Maryland); Nancy Craig (Johns Hopkins School of Medicine).
TARGET AUDIENCES: The scientific community interested in the genetic control of insects, the IAEA-FAO, extension officers who would implement genetic control practices in the field, citizens who are affected by vector-borne disease; mosquito abatement district officers in California.
Impacts
The impacts of our research over the past 12 months have been demonstration to those in the field that the beta2 tubulin promoter of Aedes aegypti can drive male specific expression in mosquitoes and so may be able to be used as a sex-specific genetic marker; the demonstration that excision assays in yeast can be efficiently used to identify null and hyperactive mutants of transposases; the demonstration that RNAi can used in Culex species; the completion of the sequencing of the Culex pipiens quinquefasciatus genome (and its distribution on the internet at NCBI).
Publications
- Subramanian, R. A., P. Arensburger, P. W. Atkinson, D. A. O'Brochta (2007). Transposable element dyanamics of the hAT element Herves in the human malaria vector Anopheles gambiae ss. Genetics 176: 2477-2487. Atkinson, P. W., D. A. OBrochta and N. L. Craig (2007). The hobo, Hermes and Herves Transposable Elements of Insects. In Vreyson, M. J. B., A. S. Robinson, and J. Hendichs (Eds.), Area-Wide Control of Insect Pests: From Research to Field Implementation. Springer, Dordrecht, The Netherlands (in press). Nene, V., J. R. Wortman, D. Lawson, B. Haas, C. Kodira, Z. Tu, B. Loftus, Z. Xi, K. Megy, M. Grabherr, Q. Ren, E. M. Zdobnov, N. F. Lobo, K. S. Campbell, S. E. Brown, M. F. Bonaldo, J. Zhu, S. P. Sinkins et al., (2007). Genome sequence of Aedes aegypti, a major arbovirus vector. Science 316:1718-1723. Lawson, D., P. Arensburger, P. Atkinson, N. J. Besansky, R. V. Bruggner, R. Butler, K. S. Campbell, G. K. Christophides, S. Christley, E. Dialynas, D. Emmert, M.
Hammond, C. A. Hill, R. C. Kennedy, N
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Progress 01/01/06 to 12/31/06
Outputs
In the past five years we have continued our identification and isolation of active transposons from mosquitoes and have used a range of biochemical techniques to map the binding sites of the Hermes and Herves transposases to the left and right ends of their respective transpososons. We have shown that the Hermes transposase autoregulates its own expression by binding to a distal rather than proximal motif. We have shown that for both Hermes and Herves, transposase binding is asymmetrical with respect to both ends, consistent with genetic data. We have isolated and identified several host factors that interacts specifically with the Hermes transposon with one actually decreasing transposition most likely through controlling access to the transpososome. We have commenced screening for Hermes and Herves hyperactive mutants using a yeast based system and have shown that the beta-2 tubulin promoter of Aedes aegypti specifically directs expression of transgenes to the
testes. We have shown, with Shou-wei Ding, that insects also have RNAi systems that are involved in anti-viral defense and have shown that at least two insect viruses have a suppressor system based on the B2 suppressor. With Nancy L. Craig we have shown the chemical mechanism of Hermes transposition and discovered that is related in both function and sequence to the V(D)J recombination system of vertebrates. These studies paved the way for the study of the crystal structure of the Hermes transposase which is the first eukaryotic transposase so far analyzed in this way. Our work on Hermes and other hAT transposons is now at the forefront of modern transposon research both within and outside of insects.
Impacts
The short-term impact on agriculture will most likely be through both a) our development of testes-specific gene expression systems in insects which drive the expression of any desired transgene only in testes. Exploitation of this system in insect species which are agricultural pests and are the subject of current sterile insect technique programs will lead to the development of genetic sexing strategies which should eliminate females from mass rearing and so make the process more cost effective, and b) through understanding the interactions of the transpositional mechanism with the nuclear machinery of the host develop rational risk assessment estimates of transposon and transgene-linked behavior in these species. These estimates should assist with rational assessments of the value of transgenic based insect genetic control strategies. The long-term benefit will build on this and will be an extension of these technologies, together with estimates of risk based on
data, into other insects as well as the development of transposon-based gene drive technologies for use in medical entomology and vector control.
Publications
- Atkinson, P. W., D. A. OBrochta and N. L. Craig (2007). The hobo, Hermes and Herves Transposable Elements of Insects. In Vreyson, M. J. B., A. S. Robinson, and J. Hendichs (Eds.), Are-Wide Control of Insect Pests: From Research to Field Implementation. Springer, Dordrecht, The Netherlands (in press).
- Lawson, D., P. Arensburger, P. Atkinson, N. J. Besansky, R. V. Bruggner, R. Butler, K. S. Campbell, G. K. Christophides, S. Christley, E. Dialynas, D. Emmert, M. Hammond, C. A. Hill, R. C. Kennedy, N. F. Lobo, M. R. MacCallum, G. Madey, K. Megy, K. Redmond, S. Russo, D. W. Severson, E. O. Stinson, P. Topalis, E. M. Zdobnov, E. Birney, W. M. Gelbart, F. C. Kafatos, C. Louis, and F. H. Collins. (2007). VectorBase: a home for invertebrate vectors of human pathogens. Nucl. Acids. Res. (in press).
- Smith, R. C., M. F. Walter, R. H. Hice, D. A. OBrochta and P. W. Atkinson (2007). Testes-specific expression of the beta 2 tubulin promoter of Aedes aegypti and its application as a genetic sex-separation marker. Insect Mol. Biol. 16: 61-71.
- OBrochta, D. A., Subramanian, R. A., Orsetti, J., Peckham, E., Nolan, N., Arensburger, P., Atkinson, P. W. and J. D. Charlwood. (2006). hAT element population genetics in Anopheles gambiae s.l. in Mozambique. Genetica 127:185-198.
- Arensberger,P., Y-J Kim, J. Orsetti, C. Aluvihare, D. A. OBrochta and P. W. Atkinson. (2005). An active transposable element, Herves, from the African malaria Plasmodium. Genetics. 169:697-708.
- Zhou, L., R. Mitra, P. W. Atkinson, A B. Hickman, F. Dyda and N. L. Craig. (2004). Transposition of hAT elements links transposable elements and V(D)J recombination. Nature 432:995-1001.
- Li. W. X., H. Lu, R. Lu, F. Li, M. Dus, P. Atkinson, E. W. Brydon, K. L. Johnson, A. Garcia-Sastre, L. A. Ball, P. Palese, and S. W. Ding. (2004). Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing. Proc. Natl. Acad. Sci. USA 101:11350-1355.
- Atkinson, P. W. Transgenic Mosquitoes and DNA Research Safeguards. In: The Biology of Disease Vectors (2nd edition). (B. J. Beaty and W. C. Marquardt, eds.). Accepted by Elsevier Press May, 2004. Atkinson, P. W., D. A. OBrochta, and A. S. Robinson. Insect Transformation of Use in Control. In: Comprehensive Insect Physiology, Pharmacology and Molecular Biology. (L. I. Gilbert, K. Iatrou, and S. S. Gill, eds.). Accepted by Elsevier Press July, 2004. 22 ms pp.
- Robinson, A. S., G. Franz and P. W. Atkinson. (2004). Insect transgenesis and its potential role in agriculture and human health. Insect Biochem. Mol. Biol. 34:113-120.
- Wang, X. H., R. Aliyari, . X. Li, H. W. Li, K. Kim, R. Carthew, P. Atkinson, and S. W. Ding (2006). RNA interference directs innate immunity in viruses in adult Drosophila. Science 312:452-454. Atkinson, P. W. (2005). Green light for mosquito control. Nature Biotech.23:1371-1372.
- Atkinson, P. W. and D. A. OBrochta. 2004. Transgenic Malaria. Society for General Microbiology Symposium. Microbe Vector Interactions in Vector Borne Diseases. University of Bath, UK, Mar. 29-Apr. 2, 2004. Cambridge University Press. 63: 345-362.
- Rowan, K., Orsetti, Atkinson, P.W., and D. A. OBrochta. (2004). Tn5 as an insect gene vector. Insect Bioch. Molec. Biol. 34:695-705.
- Irvin, N., M. S. Hoddle, D. A. OBrochta, B. Carey and P. W. Atkinson. (2004). Assessing fitness costs for transgenic Aedes aegypti expressing the green fluorescent protein marker and transposase genes. Proc. Natl. Acad. Sci. 1001:891-896.
- OBrochta DA, Sethuraman N, Wilson R, Hice RH, Pinkerton AC, LeVesque CS, Bideshi DK, Jasinskiene N, Coates CJ, James AA, Lehane MJ, Atkinson PW (2003) Gene vector and transposable element behavior in mosquitoes. J. Exp. Biol. 206:3823-3834.
- Michel. K. and Atkinson, P. W. (2003). Nuclear localization of the Hermes transposase depends on basic amino acid residues at the N-terminus of the protein. J. Cell. Biochem. 89:778-790.
- Michel, K., OBrochta, D. A and Atkinson, P. W.. (2003) The C-terminus of the Hermes transposase contains a protein dimerization domain. Insect Biochem. Molec. Biol. 33:959-970
- Atkinson, P. W. and D. A. OBrochta. 2003. Genetic Engineering of Insects. In: Encyclopedia Insects. (R. T. Carde and V. H. Resh, eds.). Academic Press pp. 471-488.
- Wilson, R., Orsetti, J., Klocko, A.D., Aluvihare, C., Peckham, E., Atkinson, P.W., Lehane, M.J. and D.A. OBrochta. (2003). Post-integration behavior of a mariner gene vector in Aedes aegypti. Insect Biochem. Molec. Biol. 33:853-863.
- Guimond, N., D. K. Bideshi, A. C. Pinkerton, P.W. Atkinson and D. A. OBrochta. (2003). Patterns of Hermes transposition in Drosophila melanogaster. Mol. Gen. Genomics. 268:779-790.
- Holt RA, Subramanian GM, Halpern A, et al. (2002). The genome sequence of the malaria mosquito Anopheles gambiae. Science. 298:129-49.
- Michel, K., D. A. OBrochta and P. W. Atkinson. (2002). Does the DES motif form the active center in the Hermes transposase? Gene.29:141-146
- Atkinson, P. W., and K. Michel. (2002). Whats buzzing? Mosquito genomics and transgenic mosquitoes. Genesis. 32:42-48.
- Atkinson, P. W. and A. A. James. 2002. Germline Transformants Spreading Out to Many Insect Species. Advances in Genetics 47: 49-86.
- Atkinson, P. W. (2002). Genetic engineering in insects of agricultural importance. Insect. Biochem. Mol. Biol. 32:1237-1242.
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Progress 01/01/05 to 12/31/05
Outputs
In 2005 we achieved several breakthroughs. We published our discovery of the Herves transposon from the malaria mosquito Anopheles gambiae and commenced a mutagenesis assay of this element. We demonstrated that this transposon is active in yeast thereby allowing us to develop high throughput genetic screens in this organism for hyperactive and hypoactive forms of the transposase. These complement our studies on the related Hermes transposon from the housefly in which we are already testing some candidate hyperactive mutants in insects. These studies have been augmented by the availability of the crystal structure of the Hermes transposase. This provides important insights into how this enzyme may function and so enables us to target specific amino acid residues and then determine the impact that changing these has on enzyme function. We discovered three subfamilies of hAT transposons in the genome of the mosquito Aedes aegypti which is the principle vector of dengue
and yellow fever. These are related to the Hermes and Herves elements described above. These elements were discovered using bioinformatics and all are predicted to be functional. One of these subfamilies is particularly interesting and we have cloned one member of it and indeed shown it to be functional. This transposon, called AeBuster1, is closely related to a gene in humans, called Buster, the function of which is unknown. Buster itself is highly conserved in many mammals pointing to it being under strong positive selection. The significance of the Buster discovery is twofold. First it is the first active transposon discovered in Ae. aegypti. This mosquito is a prime target for genetic control strategies and an active endogenous element may well provide a means by which high frequency genetic transformation of this species can be routinely obtained. Furthermore this transposon may be able to drive beneficial genes through Aedes populations. To this end we are constructing AeBuster1
transposons for Aedes transformation. Second, the relationship between this Buster transposase and the human homolog demands investigation of the function of the human gene and whether it has a function in recombination and chromosome stability. It also provides us with an important model for examining how possible horizontal transfer between mosquitoes and humans may or may not occur and what impact this may have on genetic control programs. Finally in 2005 we are able to identify a number of insect genes that may be host factor genes controlling Hermes and Herves mobility insects. The identity of these genes is important since they may influence the behavior of transposons in new hosts and so directly affect the outcome of any genetic control program. We have identified a choline kinase-like gene in Drosophila that we believe acts by phosophorylating the transposase leading to a conformation change that may act as a regulatory switch. Interestingly these kinases are potent oncogenes
in mammals perhaps suggesting a link between transposon regulation and oncogenesis.
Impacts
The long-term goal of our research is to develop viable and robust gene transfer technologies for insects of economic and medical importance and in doing so bring the full repertoire of contemporary genetics to bear on relevant problems in entomology for the benefit of the citizens of California. The central bulwark of our research is that effective genetic control programs based on the use of transposable elements to transform insects can only be developed if we understand how these elements function both in vitro and in vivo. Our program is unique amongst those who use transposons in entomology since we use the tools of biochemistry and molecular genetics to understand how these transposons function. Our program is unique amongst transposable element biologists in that we seek to understand how these transposons function in different hosts. The impact of our work will be a sufficient level of understanding of these transposons which will enable their efficient use
in genetic control strategies aimed particularly at insects that vector human disease, such as mosquitoes. Our work will enable sensible and realistic estimates to be made of any risk associated with this type of genetic control since the calculus we will employ will be based on empirical measurements of transposon behavior with knowledge of the chemical basis of transposition. These outcomes will greatly facilitate the deployment of this technology in the field and increase the probability that a favorable outcome will be achieved.
Publications
- O'Brochta, D. A., Subramanian, R. A., Orsetti, J., Peckham, E., Nolan, N., Arensburger, P., Atkinson, P. W. and J. D. Charlwood. 2005. hAT element population genetics in Anopheles gambiae s.l. in Mozambique. Genetica. (in press)
- Atkinson, P. W. (2005). Green light for mosquito control. Nature Biotech.23:1371-1372.
- Arensberger,P., Y-J Kim, J. Orsetti, C. Aluvihare, D. A. O'Brochta and P. W. Atkinson. (2005). An active transposable element, Herves, from the African malaria Plasmodium. Genetics. 169:697-708.
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Progress 01/01/04 to 12/31/04
Outputs
1. We have shown that transposable elements placed into the mosquito Aedes aegypti through the process of genetic transformation become relatively immobile. This is consistent with the post-integration behavior of the mariner transposable element in transgenic Drosophila. The mechanism by which this inactivation occurs is unknown. It is not thought to be due to integration into heterochromatin since several of the transposable elements have clearly integrated into euchromatic DNA. While inactivation of movement is ideal for the construction of stable genetic strains, subsequent movement of these elements is required for several genetic control strategies envisaged for mosquitoes that are based on population replacement. The basis of this inactivation remains to be investigated. One possibility is localized silencing of the incumbent transposase either through methylation or through an RNA-mediated silencing pathway. 2. We have shown that transgenesis of mosquitoes can
come with a cost of genetic fitness. This is of concern for all insect genetic control programs since insect genotypes generated for the purpose of genetic control need to efficiently compete with field insects in order to pass their genes into subsequent generations. This is critical for population replacement strategies and even for sterile insect technique strategies in which the released insects must be competitive upon release. Our studies used established parameters for measuring fitness and applied them to several transgenic lines of mosquitoes. The decrease in fitness observed in all of them indicates that, at least in these lines, there is a fitness cost to transgenesis. The magnitude of this cost needs to be overcome in order for insect genetic control strategies using transposable elements to be effective. 3. We identified, isolated and characterized a new transposable element, Herves, from the malaria mosquito, Anopheles gambiae. Herves is functional, as shown by the
ability to use this as a gene vector in Drosophila, and is the first functional class II transposable element isolated from this important mosquito vector of malaria. Herves may well prove to be useful in efforts to develop gene vectors in Anopheline vectors. Preliminary studies show that Herves is present in field populations of Anopheles in Africa and is in disequilibrium in them, suggesting it is currently active. 4. We participated in the determining that the Hermes transposable element excises and transposases via a mechanism that is very similar to that used by the human Rag recombinases in the generation of immunoglobulin genes and T cell receptor genes in developing B and T lymphocytes. This links extant transposable elements with this process of V(D)J recombination and so opens up the possibility of using these insect transposable elements to study aspects of this vertebrate recombination pathway required for the development of the acquired immune system. Interestingly there
is evidence that aberrations in V(D)J recombination can lead to the development of some blood cancers.
Impacts
Our work on insect transposable elements used for the transformation of economically and medically important insects provides a sound basis for the future application of genetics to contemporary problems in agriculture and medicine. These will not rely on the use of chemical or viral insecticides and will be specific to the target species. Our goal is to develop these transposable elements as gene vectors by understanding how they function both in the insect and in vitro. Our studies in 2004 have increased our understanding of these elements through linking them with the important process of vertebrate V(D)J recombination. This has enabled us to extrapolate between these this recombination process and transposition and, in doing so, we have gained insights into the function of Hermes in insects. In addition our bioinformatics studies have enabled us to isolate the first functional transposable element from a mosquito species. This transposable element may well form
the basis of future genetic control strategies in mosquitoes.
Publications
- O'Brochta, D. A., N. Sethuramuran, R. Wilson, R. H. Hice, A. C. Pinkerton, C. S. Levesque, D. K. Bideshi, N. Jasinskiene, C. J. Coates, A. A. James, M. J. Lehane, and P. W. Atkinson. 2004. Gene Vector and Transposable Element Behavior in Mosquitoes. J. Exp. Biol. 206: 3823-3834.
- Arensburger, P., Y.-J. Kim, J. Orsetti, C. Aluvihare, D. A. O'Brochta, and P. W. Atkinson. 2005 Herves, An Active Transposable Element from the African Malaria Mosquito, Anopheles gambiae. Genetics, published on line, November 2004.
- Zhou, L., R. Mitra, P. W. Atkinson, A. Burgess Hickman, F. Dyda, and N. L. Craig. 2004 hAT Element Transposition Directly Links Transposable Elements and V(D)J Recombination. Nature 432: 995-1001.
- Rowan, K. H., J. Orsetti, P. W. Atkinson, and D. A. O'Brochta. 2004. Tn5 as an Insect Gene Vector. Insect Biochem. and Molec. Biol. 34: 695-705.
- Irvin, N. A., M. S. Hoddle, D. A. O'Brochta, B. Carey, and P. W. Atkinson. 2004. Assessing Fitness Costs for Transgenic Aedes aegypti Expressing the Green Fluorescent Protein Marker and Transposase Gene. Proc. Natl. Acad. Sci. 101: 891-896.
- Robinson, A. S., G. Franz, and P. W. Atkinson. 2004. Insect Transgenesis and Its Potential Role in Agriculture and Human Health. Insect Biochem. Molec. Biol. 34: 113-120.
- Li, W.-X., H. Li, R. Lu, F. Li, P. Atkinson, M. Dus, P. Atkinson, K. L. Johnson, A. Gracia-Sastre, E. Brydon, A. Ball, P. Palese, and S.-W. Ding. 2004. Interferon Antagonist Proteins of Influenza and Vacinnia Viruses are Suppressors of RNA Silencing. Proc. Natl. Acad. Sci., USA 101: 1350-1355.
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Progress 01/01/03 to 12/31/03
Outputs
We have achieved significant progress during 2003. First we have shown that transgenic lines of the yellow fever and dengue-transmitting mosquito, Aedes aegypti, are significantly less fit than their conspecific, non-transformed counterparts. These data are the first to directly measure components of fitness in transgenic mosquitoes and, in doing so, illustrate the need to quantitate these parameters in transgenic insects that are generated for the purposes of genetic control. In 2003 we also identified, isolated and characterized a new transposable element, Herves, from the malaria mosquito, Anopheles gambiae. Herves was initially identified in silico and then cloned from genomic DNA from this species. We have shown that Herves is a functional element and, as such, is the first functional element identified from any mosquito species. We have also shown that this element is possibly still active in field populations of Anopheles and that it also displays slightly
different mobility properties to the related Hermes and hobo elements. Herves may well provide an illustration of how active transposable elements can move through wild insect populations and so may well serve to bridge the gap between field behavior and laboratory design of genetic control experiments. We have also recently identified part of what we believe to be a related transposable element from Aedes aegypti. In very related, an ongoing, work, we continue to investigate the structure:function relationships of the Hermes element with the aim being to improve its use as an agent in the genetic control of insect pest species. Results of these studies will be applied to the Herves element.
Impacts
We are seeking to enhance genetic control strategies for the control and/or eradication of insect pest species. Genetic control strategies are environmentally benign and sustainable. Applied to the vectors of human disease, they should be effective, based on previous strategies that have targeted the vector species. The outcome of the successful application of these technologies will be sustainable agricultural practices with little or no environmental cost and the control of vector borne (for example mosquitoes) human disease.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
Work continues on the feasibility of genetic control strategies in insects as well as developing genetic tools for exploring insect genomes. We have continued experiments aimed at developing enhancer trap technologies for mosquito species, and have continued our examination of the structure: function relationships of two transposable elements - Hermes and hobo - that are used to genetically transform insects with the aim being to develop improved transposable element gene vectors. Work continues on the investigation into the ability of the Hermes element to spread through caged populations of fruit fly and mosquito and, in collaboration with Dr. Mark S. Hoddle, we are investigating the fitness consequences of transgenesis on mosquitoes. We have also been able to rear olive fly in the laboratory in preparation for a feasibility study on determining whether a sterile insect technique program can be established for this pest insect in southern California.
Impacts
Genetic transformation techniques continue to be developed and improved for insect species that are of medical and agricultural importance to California and the United States.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
In the past 12 months we have continued a multifaceted approach into exploring the feasibility of genetic control strategies in insects as well as developing genetic tools for exploring insect genomes. Specifically we have commenced experiments aimed at developing enhancer trap technologies for mosquito species, have continued our examination of the structure: function relationships of two transposable elements - Hermes and hobo - that are used to genetically transform insects with the aim being to develop improved transposable element gene vectors. We have commenced an investigation into the ability of the Hermes element to spread through caged populations of fruit fly and mosquito and, in collaboration with Dr. Mark S. Hoddle, we have commenced an investigation into the fitness consequences of transgenesis on mosquitoes. We have also been able to rear olive fly in the laboratory in preparation for a feasibility study on determining whether a sterile insect technique
program can be established for this pest insect in southern California.
Impacts
We continue to develop and improve genetic transformation techniques for insect species that are of medical and agricultural importance to California and the United States. These techniques will enable the genetic dissection of biochemical pathways in these insect species and the knowledge gained from this will lead to new approaches to insect pest control. They will also enable new genetic strains to be generated that may be directly used for the genetic control of pest insects and/or the diseases they vector.
Publications
- Allen, M. L.,.O'Brochta, D. A, Atkinson, P.W. and LeVesque C. S. (2001) Stable germ-line transformation of Culex quinquefasciatus (Diptera: Culicidae). Journal of Medical Entomology 38, 701-710.
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Progress 01/02/00 to 12/31/00
Outputs
In the past year we have further demonstrated that the Hermes transposable element can act as a gene vector in the mosquitoes Aedes aegypti and Culex quinquefasciatus and also shown that it can genetically transform the Mediterranean fruit fly, Ceratitis capitata and the stable fly, Stomoxys calcitrans. Medfly is an extremely important pest to California while the stable fly is a pest of livestock in this state. Establishment of these technologies in these important pests allows for the development of new genetic based strategies for pest control. Our ongoing work in the mosquito species has focused on two aspects. The first is to determine if autonomous Hermes elements can spread through caged populations of Ae. aegypti. This is an important question since transposable elements like Hermes have been proposed to be genetic drive agents capable of spreading genes through populations. The second aspects focuses on developing a transgenic strain of Culex quinquefasciatus
that is incapable of transmitting filiarial worms - the agents of filariasis. To this end we have generated a transgenic line of Culex that has targeted gene expression in a tissue required for the development of filarial worms in the mosquito.
Impacts
We have developed genetic transformation techniques for insect species of medical and agricultural importance to California and the United States. These techniques are of significance for two reasons. First, they enable the genetic dissection of biochemical pathways in these insect species and the knowledge gained from this will lead to new approaches to pest control. Second, they permit the development of new strains of insects that are refractory to the spread of insect vector-borne disease
Publications
- Morgan, D. J. W., S. R. Reitz, P.W. Atkinson and J. T. Trumble. 2000. The resolution of Californian populations of Liriomyza huidobrensis and Liriomyza triflii (Diptera: Agromyzidae) using PCR. Heredity. 85, 53-61.
- O'Brochta, D. A., P. W. Atkinson and M. J. Lehane. 2000. Transformation of Stomoxys calcitrans with a Hermes gene vector. Insect Mol. Biology. 9, 531-538.
- Atkinson, P. W. and D. A. O'Brochta. 2000. Hermes and other hAT elements as gene vectors in insects. In: Insect Transgenesis: Methods and Application (A. A. James and A. M. Handler, eds.). CRC Press, Boca Raton, FL. pp. 219-235.
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Progress 01/01/99 to 12/31/99
Outputs
In the past year we have demonstrated that the Hermes transposable element can act as a gene vector in the mosquitoes Aedes aegypti and Culex quinquefasciatus. Both are important vectors of disease, Culex in particular can be a vector of human and livestock disease in California. During the course of these studies we also demonstrated that the green fluorescent protein gene can be efficiently used as a genetic marker in mosquitoes. We also used the Hermes element to genetically transform the Mediterranean fruit fly, C. capitata, thereby increasing our ability to develop sophisticated genetic technologies in this important pest species. To this end, we have succeeded in the isolation and characterization of a gene, called transformer-2, involved in the sexual development of the Mediterranean fruit fly. Introduction of modified forms of this into the C. capitata genome should enable new approaches to the genetic control of this insect to be developed.
Impacts
We have developed genetic transformation techniques for insect species of medical and agricultural importance to California and the United States. These techniques are of significance for two reasons. First, they enable the genetic dissection of biochemical pathways in these insect species and the knowledge gained from this will lead to new approaches to pest control. Second, they permit the development of new strains of insects that are refractory to the spread of insect vector-borne disease.
Publications
- Pinkerton, A. C., K. Michel, D. A. O'Brochta and P. W. Atkinson, (2000). Green fluorescent protein as a genetic marker in transgenic Aedes aegypti. Insect Molec. Biol .9: 1-10.
- Saville, K. J., W. D. Warren, P. W. Atkinson and D. A. O'Brochta (1999). Integration specificity of the hobo element of Drosophila melanogaster is dependent on sequences flanking the target site. Genetica.105: 133-147.
- Atkinson, P. W. and D. A. O'Brochta, (1999). Genetic transformation of non-drosophilid insects by transposable elements. Annals of the Entomological Society of America 92: 930-936.
- Pinkerton, A. C., S. Whyard, H. A. Mende, C. J. Coates, D. A. O'Brochta and P. W. Atkinson, (1999). The Queensland fruit fly, Bactrocera tryoni, contains multiple members of the hAT family of transposable elements. Insect Molec. Biol. 8: 423-434.
- Sundararajan, P., P. W. Atkinson and D. A. O'Brochta, (1999). Transposable element interactions in insects: Crossmobilization of Hermes and hobo. Insect Molec. Biol.359 -368
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Progress 01/01/98 to 12/01/98
Outputs
The two objectives of this project are: To develop gene vectors that can be used to efficiently genetically transform insects of economic importance; To develop genetic markers that will enable the efficient identification of transgenic insects both in the laboratory and in the field. We have made progress in both of these. We have used the Hermes transposable element from the house fly, Musca domestics, to genetically transform three species of insect - the vinegar fly, Drosophila melanogaster, and two significant pest species, the Mediterranean fruit fly, Ceratitis capitata and the yellow fever mosquito, Aedes aegypti. Significantly transformants of Ae. aegypti and D. melanogaster were recognized using the green fluorescent protein from the jellyfish, Aequorea victoria. This was the first time this genetic marker had been used to recognize genetic transformants of any mosquito species. Moreover a wild-type strain of Ae. aegypti was transformed. This will enable
entomologists and parasitologists to develop genetic strategies in which wild-type strains of mosquitoes can be used as the recipient and so will expand our ability to genetically manipulate this species in order to develop new strategies for the genetic control of this species. Our ability to genetically transform the medfly, C. capitata, with the Hermes element now provides researchers with three transposable elements system to use in this commercially important species. This now places medfly geneticists in the position be being able to develop powerful genetic tools, such as gene tagging and enhancer trapping,.
Impacts
(N/A)
Publications
- PINKERTON, A. C., MICHEL, K., O'BROCHTA, D. A., ATKINSON, P. W. Green Fluorescent Protein as a Genetic Marker in Transgenic Aedes aegypti. Insect Molecular Biology (submitted).
- PINKERTON, A. C., WHYARD, S., MENDE, H. M., COATES, C. J., O'BROCHTA, D. A. and ATKINSON, P. W. The Queensland fruit fly, Bactrocera tryoni, contains multiple members of the hAT family of transposable
- SUNDARARAJAN, P., ATKINSON, P. W. and O'BROCHTA, D. A. Transposable element interactions in insects: Crossmobilization of Hermes and hobo. Insect Molecular Biology (In press).
- SAVILLE, K. J., WARREN, W., ATKINSON, P. W., and O'BROCHTA, D. A. Target site specificity of the hobo transposable element of Drosophila melanogaster. Insect Molecular Biology (In press).
- LEOPOLD, R.A. and ATKINSON, P.W. 1999. Cryopreservation of sheep blow fly embryos, Lucilia cuprina (Diptera: Calliphoridae). Cryo-lett. (In
- ATKINSON, P. W. and O'BROCHTA, D. A. Genetic transformation of non-drosophilid insects by transposable elements. Annals of the Entomological Society of America (In press).
- O'BROCHTA, D. A. and ATKINSON, P. W. (1998). Building the Better bug. Scientific American, December: 60-65.
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