Germ-line transformation via transposable elements is a powerful tool to study gene function in Drosophila melanogaster. However, some inherent characteristics of transposon-mediated transgenesis limit its use for transgene analysis. Here, we circumvent these limitations by optimizing a C31-based integration system. We generated a collection of lines with precisely mapped attP sites that allow the insertion of transgenes into many different predetermined intergenic locations throughout the fly genome. By using regulatory elements of the nanos and vasa genes, we established endogenous sources of the C31 integrase, eliminating the difficulties of coinjecting integrase mRNA and raising the transformation efficiency. Moreover, to discriminate between specific and rare nonspecific integration events, a white gene-based reconstitution system was generated that enables visual selection for precise attP targeting. Finally, we demonstrate that our chromosomal attP sites can be modified in situ, extending their scope while retaining their properties as landing sites. The efficiency, ease-of-use, and versatility obtained here with the C31-based integration system represents an important advance in transgenesis and opens up the possibility of systematic, highthroughput screening of large cDNA sets and regulatory elements.attP landing sites ͉ germ-line transformation ͉ site-specific integration A major goal in the present era of genomics is to identify and functionally characterize all genes relevant to a specific pathway or biological process. With its powerful repertoire of genetic tools, the multicellular model organism Drosophila melanogaster has played an eminent role in this endeavor (1). One method to identify relevant genes is to perform chemical mutagenesis screens of various kinds. Another very fruitful approach in Drosophila has been the use of P-element-mediated germ-line transformation (2, 3), especially when combined with tools such as the Gal4/UAS expression system (4) or when it is used for insertional mutagenesis (5, 6). One characteristic of P-elements is their random integration behavior. Although this ''randomness'' is advantageous for generating mutations and deletions, it is generally not ideal for transgene analysis. The random integration of P-elements necessitates considerable effort to map insertions. Genomic position effects complicate the analysis of transgenes and render precise structure/ function analyses nearly impossible. A further shortcoming of the P-element system is its relatively moderate transformation efficiency, a significant hurdle to any large-scale transgenesis effort.Strategies have been developed to circumvent the problem of randomness by targeted integration systems in Drosophila, which are generally based on the FLP and Cre recombinases (7-9, 32). Such techniques permit precise targeting to genomic landing sites but are still handicapped by transformation rates that are, at best, moderately higher than those achieved with the P-element system (8). Furthermore, especially for FL...

As one of two Drosophila Hox clusters, the bithorax complex (BX-C)is responsible for determining the posterior thorax and each abdominal segment of the fly. Through the dissection of its large cis-regulatory region,biologists have obtained a wealth of knowledge that has informed our understanding of gene expression, chromatin dynamics and gene evolution. This primer attempts to distill and explain our current knowledge about this classic, complex locus.

The acetylcholine receptor from vertebrate skeletal muscle is a pentamer of homologous subunits with composition alpha 2 beta gamma delta. Its two ligand binding sites, formed at alpha-gamma and alpha-delta interfaces, differ in their affinities for agonists and competitive antagonists, owing to different contributions of the gamma and delta subunits. To identify portions of the gamma and delta subunits that contribute to the binding sites, the experiments described here use gamma-delta subunit chimeras and site-specific mutants to determine the basis of the 10,000-fold selectivity of conotoxin M1 for the sites. Three distinct regions of the extracellular domain were found to contribute to conotoxin M1 selectivity, each containing a single residue responsible for the contribution of that region. Residues K34, S111, and F172 of the gamma subunit confer low affinity to the alpha-gamma binding site, whereas the corresponding residues of the delta subunit, S36, Y113, and I178, confer high affinity to the alpha-delta site. Identification of three separate determinants of ligand selectivity suggests a limited model of the folding pattern of the extracellular domain of the subunits.

In insects, products of the male reproductive tract are essential for initiating and maintaining the female post-mating response (PMR). The PMR includes changes in egg laying, receptivity to courting males, and sperm storage. In Drosophila, previous studies have determined that the main cells of the male accessory gland produce some of the products required for these processes. However, nothing was known about the contribution of the gland's other secretory cell type, the secondary cells. In the course of investigating the late functions of the homeotic gene, Abdominal-B (Abd-B), we discovered that Abd-B is specifically expressed in the secondary cells of the Drosophila male accessory gland. Using an Abd-B BAC reporter coupled with a collection of genetic deletions, we discovered an enhancer from the iab-6 regulatory domain that is responsible for Abd-B expression in these cells and that apparently works independently from the segmentally regulated chromatin domains of the bithorax complex. Removal of this enhancer results in visible morphological defects in the secondary cells. We determined that mates of iab-6 mutant males show defects in long-term egg laying and suppression of receptivity, and that products of the secondary cells are influential during sperm competition. Many of these phenotypes seem to be caused by a defect in the storage and gradual release of sex peptide in female mates of iab-6 mutant males. We also found that Abd-B expression in the secondary cells contributes to glycosylation of at least three accessory gland proteins: ovulin (Acp26Aa), CG1656, and CG1652. Our results demonstrate that long-term post-mating changes observed in mated females are not solely induced by main cell secretions, as previously believed, but that secondary cells also play an important role in male fertility by extending the female PMR. Overall, these discoveries provide new insights into how these two cell types cooperate to produce and maintain a robust female PMR.

Histone chaperones are involved in a variety of chromatin transactions. By a proteomics survey, we identified the interaction networks of histone chaperones ASF1, CAF1, HIRA, and NAP1. Here, we analyzed the cooperation of H3/H4 chaperone ASF1 and H2A/H2B chaperone NAP1 with two closely related silencing complexes: LAF and RLAF. NAP1 binds RPD3 and LID-associated factors (RLAF) comprising histone deacetylase RPD3, histone H3K4 demethylase LID/KDM5, SIN3A, PF1, EMSY, and MRG15. ASF1 binds LAF, a similar complex lacking RPD3. ASF1 and NAP1 link, respectively, LAF and RLAF to the DNA-binding Su(H)/Hairless complex, which targets the E(spl) NOTCH-regulated genes. ASF1 facilitates gene-selective removal of the H3K4me3 mark by LAF but has no effect on H3 deacetylation. NAP1 directs high nucleosome density near E(spl) control elements and mediates both H3 deacetylation and H3K4me3 demethylation by RLAF. We conclude that histone chaperones ASF1 and NAP1 differentially modulate local chromatin structure during gene-selective silencing.