Position effects can complicate transgene analyses. This is especially true when comparing transgenes that have inserted randomly into different genomic positions and are therefore subject to varying position effects. Here, we introduce a method for the precise targeting of transgenic constructs to predetermined genomic sites in Drosophila using the fC31 integrase system in conjunction with recombinase-mediated cassette exchange (RMCE). We demonstrate the feasibility of this system using two donor cassettes, one carrying the yellow gene and the other carrying GFP. At all four genomic sites tested, we observed exchange of donor cassettes with an integrated target cassette carrying the mini-white gene. Furthermore, because RMCE-mediated integration of the donor cassette is necessarily accompanied by loss of the target cassette, we were able to identify integrants simply by the loss of mini-white eye color. Importantly, this feature of the technology will permit integration of unmarked constructs into Drosophila, even those lacking functional genes. Thus, fC31 integrase-mediated RMCE should greatly facilitate transgene analysis as well as permit new experimental designs. B IOLOGICAL research is greatly facilitated by our ability to manipulate DNA sequences in vivo. In the model organism Drosophila melanogaster, exogenous sequences are routinely incorporated into the genome using the P-element transposon system developed by Rubin and Spradling (Rubin and Spradling 1982;Spradling and Rubin 1982). The harnessing of this technology afforded a new ability to alter the genome, allowing the construction of transgenic animals by the relatively simple method of embryonic injection. Furthermore, P elements have been indispensable as a platform for developing new technologies for Drosophila research including the GAL4/UAS system (Brand and Perrimon 1993), targeted deletions (Ryder et al. 2004), and high-resolution mapping of mutations (Zhai et al. 2003).Recently, strategies have been developed to repeatedly incorporate transgenes into a single position in the genome. This provides a significant advantage over conventional P-element transformation, which occurs in an untargeted fashion and therefore subjects transgenes to varying position effects. In general these new strategies make use of site-specific recombinases, which catalyze crossovers between defined target sequences (Branda and Dymecki 2004). The most popular of these enzymes are FLP and Cre, which are widely used for many applications in Drosophila, including clonal analysis, targeted deletions, and tissue-specific excision (Golic and Lindquist 1989;Golic 1991; Hartl 1996, 2000;Heidmann and Lehner 2001;Ryder et al. 2004). A simple form of transgene targeting using these enzymes involves P elements carrying two transgenes, one flanked by loxP target sites for the Cre recombinase and the other flanked by FRT target sites for the FLP recombinase. Once such a P element integrates, subsequent treatment with Cre or FLP excises one or the other transgene, effectiv...

To identify genetic variants contributing to end-stage renal disease (ESRD) in type 2 diabetes, we performed a genome-wide analysis of 115,352 single nucleotide polymorphisms (SNPs) in pools of 105 unrelated case subjects with ESRD and 102 unrelated control subjects who have had type 2 diabetes for >10 years without macroalbuminuria. Using a sliding window statistic of ranked SNPs, we identified a 200-kb region on 8q24 harboring three SNPs showing substantial differences in allelic frequency between case and control pools. These SNPs were genotyped in individuals comprising each pool, and strong evidence for association was found with rs2720709 (P ‫؍‬ 0.000021; odds ratio 2.57 [95% CI 1.66 -3.96]), which is located in the plasmacytoma variant translocation gene PVT1. We sequenced all exons, exon-intron boundaries, and the promoter of PVT1 and identified 47 variants, 11 of which represented nonredundant markers with minor allele frequency >0.05. We subsequently genotyped these 11 variants and an additional 87 SNPs identified through public databases in 319-kb flanking rs2720709 (ϳ1 SNP/3.5 kb); 23 markers were associated with ESRD at P < 0.01. The strongest evidence for association was found for rs2648875 (P ‫؍‬ 0.0000018; 2.97 [1.90 -4.65]), which maps to intron 8 of PVT1. Together, these results suggest that PVT1 may contribute to ESRD susceptibility in diabetes. Diabetes 56: [975][976][977][978][979][980][981][982][983] 2007

Background: Pooling genomic DNA samples within clinical classes of disease followed by genotyping on whole-genome SNP microarrays, allows for rapid and inexpensive genome-wide association studies. Key to the success of these studies is the accuracy of the allelic frequency calculations, the ability to identify false-positives arising from assay variability and the ability to better resolve association signals through analysis of neighbouring SNPs.

Eukaryotic enhancers act over very long distances, yet still show remarkable specificity for their own promoter. To better understand mechanisms underlying this enhancer-promoter specificity, we used transvection to analyze enhancer choice between two promoters, one located in cis to the enhancer and the other in trans to the enhancer, at the yellow gene of Drosophila melanogaster. Previously, we demonstrated that enhancers at yellow prefer to act on the cis-linked promoter, but that mutation of core promoter elements in the cis-linked promoter releases enhancers to act in trans. Here, we address the mechanism by which these elements affect enhancer choice. We consider and explicitly test three models that are based on promoter competency, promoter pairing, and promoter identity. Through targeted gene replacement of the endogenous yellow gene, we show that competency of the cis-linked promoter is a key parameter in the cis-trans choice of an enhancer. In fact, complete replacement of the yellow promoter with both TATAcontaining and TATA-less heterologous promoters maintains enhancer action in cis.

The many reports of trans interactions between homologous as well as nonhomologous loci in a wide variety of organisms argue that such interactions play an important role in gene regulation. The yellow locus of Drosophila is especially useful for investigating the mechanisms of trans interactions due to its ability to support transvection and the relative ease with which it can be altered by targeted gene replacement. In this study, we exploit these aspects of yellow to further our understanding of cis as well as trans forms of enhancer-promoter communication. Through the analysis of yellow alleles whose promoters have been replaced with wild-type or altered promoters from other genes, we show that mutation of single core promoter elements of two of the three heterologous promoters tested can influence whether yellow enhancers act in cis or in trans. This finding parallels observations of the yellow promoter, suggesting that the manner in which trans interactions are controlled by core promoter elements describes a general mechanism. We further demonstrate that heterologous promoters themselves can be activated in trans as well as participate in pairing-mediated insulator bypass. These results highlight the potential of diverse promoters to partake in many forms of trans interactions.