The Drosophila Gene Disruption Project (GDP) has created a public collection of mutant strains containing single transposon insertions associated with different genes. These strains often disrupt gene function directly, allow production of new alleles, and have many other applications for analyzing gene function. Here we describe the addition of 7600 new strains, which were selected from .140,000 additional P or piggyBac element integrations and 12,500 newly generated insertions of the Minos transposon. These additions nearly double the size of the collection and increase the number of tagged genes to at least 9440, approximately two-thirds of all annotated protein-coding genes. We also compare the site specificity of the three major transposons used in the project. All three elements insert only rarely within many Polycomb-regulated regions, a property that may contribute to the origin of "transposon-free regions" (TFRs) in metazoan genomes. Within other genomic regions, Minos transposes essentially at random, whereas P or piggyBac elements display distinctive hotspots and coldspots. P elements, as previously shown, have a strong preference for promoters. In contrast, piggyBac site selectivity suggests that it has evolved to reduce deleterious and increase adaptive changes in host gene expression. The propensity of Minos to integrate broadly makes possible a hybrid finishing strategy for the project that will bring .95% of Drosophila genes under experimental control within their native genomic contexts.

C ytoplasmic incompatibility (CI) is the most widespread and, perhaps, the most prominent feature that Wolbachia endosymbionts impose on their hosts (1, 2). CI results in embryonic mortality (EM) in matings between insects of the same species with different Wolbachia infection status (3, 4). It can be either unidirectional or bidirectional. Unidirectional CI is typically expressed when an infected male mates with an uninfected female. The reciprocal mating is fully compatible, as are matings between infected individuals. Bidirectional CI usually occurs in matings between infected individuals harboring different strains of Wolbachia. Although the mechanism of CI has not yet been elucidated on the molecular level, several lines of evidence suggest that Wolbachia somehow modifies the paternal chromosomes during spermatogenesis (mature sperm does not contain the bacteria). This modification influences their behavior during the first mitotic divisions and results in loss of mitotic synchrony (5, 6). Even before the etiological connection between Wolbachia and CI was revealed in mosquitoes (7), attempts were made to exploit CI as a method to suppress natural populations of arthropod pests in a way analogous to the sterile insect technique (S.I.T.) (8). These early attempts involved the mass production and release of incompatible male insects to control wild populations of disease vectors such as the mosquito Culex pipiens (9) and of agricultural pests such as the European cherry fruit fly, Rhagoletis cerasi (10) and, at a smaller scale, the almond moth Cadra (Ephestia) cautella (11).The Mediterranean fruit fly (medfly) Ceratitis capitata is a worldwide pest that infests Ͼ250 fruit varieties of economic importance (12). Extensive screenings of both laboratory and natural populations of medfly have shown that this insect pest is not infected with Wolbachia (13). However, at the time of this writing, ongoing experimental work suggests that some Brazilian natural populations of medfly may be infected with Wolbachia (D. Selivon, personal communication). To determine whether the medfly can support Wolbachia infections and express CI, we used conventional embryonic cytoplasmic injections (14-17) for transfer of natural bacterial symbionts from a related species, R. cerasi (Diptera, Tephritidae) (18), to an uninfected laboratory strain of medf ly C. capitata (Diptera, Tephritidae), the Benakeion strain.Previous studies have demonstrated high levels of incompatibility between natural populations of R. cerasi (10,19), the basis of which was recently shown to be Wolbachia (18). Populations of R. cerasi are either infected by a single Wolbachia variant, wCer1, or coinfected by two variants, wCer1 and wCer2. Incompatibility occurs between males from doubly infected populations and females from singly infected populations, suggesting the wCer2 infection as the cause of CI (18). Additionally, transfer of wCer2 in Drosophila simulans also resulted to the induction of CI (17). An additional, yet uncharacterized, Wolbachia strain (wCer...

Anopheline mosquito species are obligatory vectors for human malaria, an infectious disease that affects hundreds of millions of people living in tropical and subtropical countries. The lack of a suitable gene transfer technology for these mosquitoes has hampered the molecular genetic analysis of their physiology, including the molecular interactions between the vector and the malaria parasite. Here we show that a transposon, based on the Minos element and bearing exogenous DNA, can integrate efficiently and stably into the germ line of the human malaria vector Anopheles stephensi, through a transposase-mediated process.

Exogenous functional DNA was introduced into the germline chromosomes of the Mediterranean fruit fly (medfly) Ceratitis capitata with a germline transformation system based on the transposable element Minos from Drosophila hydei. Transformants were identified as phenotypic revertants of a white-eyed mutation carried by the recipient strain. Clusters of transformants were detected among the progeny of 390 individuals screened for germline transformation. Five independent and phenotypically active integration events were identified, in each of which a single copy of the transposon was inserted into a different site of the medfly genome. Molecular analysis indicates that they represent transposase-mediated insertions of the transposon into medfly chromosomes.