Peter W. Atkinson

The Hermes transposable element from the house fly, Musca domestica , is a short inverted repeat-type element of the hobo, Ac , and Tam3 ( hAT ) element family

The genome of the house fly, Musca domestica, contains an active transposable element system, called Hermes. Using PCR and inverse PCR we amplified and sequenced overlapping segments of several Hermes elements and from these data we have constructed a 2749 bp consensus Hermes DNA sequence. Hermes termini are composed of 17 bp imperfect inverted repeats that are almost identical to the inverted terminal repeats of the hobo element of Drosophila melanogaster. Full length Hermes elements contain a single long ORF capable of encoding a protein of 612 amino acids which is 55% identical to the amino acid sequence of the hobo transposase. Comparison of the ends of the Hermes and hobo elements to those of the Ac element of Zea mays, and the Tam3 element of Antirrhinum majus, as well as several other plant and insect elements, revealed a conserved terminal sequence motif. Thus Hermes is clearly a member of the hobo, Ac and Tam3 (hAT) transposable element family, other members of which include the Tag1 element from Arabidopsis thaliana and the Bg element from Zea mays. The evolution of this class of transposable elements and the potential utility of Hermes as a genetic tool in M. domestica and related species are discussed.

Transposable elements and gene transformation in non-drosophilid insects

This review summarizes recent data on the development of non-drosophilid insect transformation systems. The discussion focuses on one particular approach to developing transformation systems that relies on the use of short inverted repeat-type transposable elements analogous to that employed for Drosophila melanogaster transformation. Representatives from four families of short inverted repeat-type transposable elements have been shown to either act as non-drosophilid gene vectors or to have the ability to transpose accurately when introduced into non-host insect cells. Minos, a member of the Tcl family of elements isolated originally from D. hydei has been successfully used as a germline transformation vector in the Medfly, Ceratitis capitata. Hermes, a member of the hAT family of elements isolated originally from Musca domestica has been successfully used as a gene transformation vector in D. melanogaster and has a host range that appears to include culicids. hobo, another member of the hAT family of elements isolated from D. melanogaster also has a broad host range that includes tephritid fruitflies. mariner(Mos), a member of the mariner family of elements isolated from D. mauritiana can transpose in calliphorids. Finally, piggyBac/IFP2, a member of the TTAA-specific family of elements isolated from Trichoplusia ni can transpose when introduced into Spodoptera frugiperda cells. Although routine transformation of insects other than D. melanogaster is not possible it is clear that the raw materials for the development of efficient transformation systems are now available.

Interplasmid transposition of the mariner transposable element in non-drosophilid insects

Abstract Plasmid-based transposition assays were performed in developing embryos of the Australian sheep blowfly Lucilia cuprina and the Queensland fruit fly Bactrocera tryoni, using the mariner transposable element from Drosophila mauritiana. Transposition products were recovered that were identical in structure to those recovered from D. melanogaster. Only sequences delimited by the mariner terminal repeats were transposed and all insertions occurred at TA residues, and duplicated these. These are the hallmarks of mariner transpositions observed in the chromosomes of D. melanogaster and D. mauritiana, indicating that the plasmid-based assays are accurate indicators of mariner transposition activity. The recovery of precise transposition products from L. cuprina and B. tryoni demonstrates that mariner should be capable of producing germline transformants in these species. The results obtained from these assays suggests that they will be extremely useful in determining if mariner can transpose in other non-drosophilid insects and for investigating factors that might affect mariner transposition frequency.

The transposable element mariner can excise in non-drosophilid insects

Plasmid-based excision assays performed in embryos of two non-drosophilid species using the mariner transposable element from Drosophila mauritiana resulted in empty excision sites identical to those observed after the excision of mariner from D. mauritiana chromosomes. In the presence of the autonomous mariner element Mos1, excision products were recovered from D. melanogaster, D. mauritiana and the blowfly Lucilia cuprina. When a hsp82 heat shock promoter-Mos1 construct was used to supply mariner transposase, excision products were also recovered from the Queensland fruitfly Bactrocera tryoni. Analysis of DNA sequences at empty excision sites led us to hypothesise that the mariner excision/repair process involves the formation of a heteroduplex at the excision breakpoint. The success of these assays suggests that they will provide a valuable tool for assessing the ability of mariner and mariner-like elements to function in non-drosophilid insects and for investigating the basic mechanisms of mariner excision and repair.

The hermit transposable element of the Australian sheep blowfly, Lucilia cuprina, belongs to the hAT family of transposable elements

We report the cloning ofhermit, a member of thehAT family of transposable elements from the genome of the Australian sheep blowfly,Lucilia cuprina. Hermit is 2716 bp long and is 49% homologous to the autonomoushobo element,HFL1, at the nucleic acid level.Hermit has 15 bp terminal inverted repeats that share 10 bp with the terminal inverted repeats ofHFL1. Conceptual translation reveals a 583 residue open reading frame (ORF) that is 64% similar and 42% identical to theHFL1 ORF. However, the sequence of thehermit element contains two frameshifts within the putative ORF, indication thathermit is an inactive element. Analysis ofL. cuprina strains from within and outside Australia suggested thathermit is present as a single copy in all the genomes analysed.

The 5' regulatory region from the Drosophila pseudoobscura hsp82 gene results in a high level of reporter gene expression in Lucilia cuprina embryos.

We have previously examined the efficiency of two Drosophila melanogaster promoters to enable reporter gene expression in embryos of the Australian sheep blowfly, Lucilia cuprina. Both the hsp70 heat-shock promoter and the actin5C promoter resulted in low levels of expression of a reporter gene in these embryos. In this study, the D. pseudoobscura hsp82 promoter (phsp82) was tested for its ability to direct the expression of the Escherichia coli chloramphenicol acetyltransferase-encoding gene (cat). We report that the level of CAT activity in L. cuprina embryos was comparable to that obtained with the same construct in D. melanogaster, indicating that phsp82 functions efficiently in this non-drosophilid insect. The results suggest that phsp82 may be utilised in other non-drosophilid insects in which poor expression levels are obtained from constructs containing the hsp70 or actin5C promoters.

The Australian bushfly Musca vetustissima contains a sequence related to transposons of the hobo, Ac and Tam3 family

Abstract The genome of the Australian bushfly Musca vetustissima contains a sequence that is highly similar to the transposase-encoding regions of the hobo, Ac and Tam3 (hAT) family of transposable elements.

An eye color gene for the detection of transgenic non-drosophilid insects

A genetic marker for identifying transgenic Musca domestica by changes in eye color is described. The Drosophila melanogaster tryptophan oxygenase gene, vermilion (v), was tested for its ability to genetically complement the mutant tryptophan oxygenase gene in houseflies homozygous for green (ge). The v cDNA, placed under the control of the hsp82 promoter of D. pseudoobscura was transiently expressed in M. domestica embryos homozygous for the tryptophan oxygenase gene, ge, resulting in the rescue of adult eye color. The use of a gene from D. melanogaster to complement an eye color mutant in Musca provides the opportunity to develop a gene vector system for M. domestica and a select group of other non-drosophilid insects in which homologous mutations exist.

In vivo expression of two highly conserved Drosophila genes in the Australian sheep blowfly, Lucilia cuprina

The expression of foreign genes in insects has often relied on the use of the promoter from the heat-shock-70 gene (hsp 70) of Drosophila melanogaster. It has been generally assumed that because this gene is highly conserved it will be expressed efficiently in other insects. We examined the efficiency of the hsp 70 promoter and the actin5C promoter from D. melanogaster in regulating gene expression in the Australian sheep blowfly, Lucilia cuprina (family: Calliphoridae). Plasmids containing these promoters 5′ of the coding region of the E. coli chloramphenical acetyltransferase (CAT) gene were microinjected into preblastoderm embryos and CAT activities subsequently measured from either developing embryos or newly hatched larvae. We report that the amount of CAT activity present in injected embryos was a function of both the plasmid copy number within the embryo and the species injected. The hsp 70 promoter was expressed 10–100 times less efficiently in L. cuprina compared to D . melanogaster and was not heat inducible, even at temperatures at which heat shock is known to occur in this insect. The actin 50 promoter was expressed approx. 10 times as efficiently in both species compared to the hsp 70 promoter. As with the hsp 70 promoter, the actin5C promoter was more efficiently expressed in D. melanogaster compared to L. cuprina. The significance of these results in relation to heterologous gene expression systems and developing germ line transformation in non-drosophilid insects is discussed.