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Insect cells are an underexplored resource for functional G-protein-coupled receptor (GPCR) assays, despite a strong record in biochemical (binding) assays. Here we describe the use of vectors capable of creating stably transformed insect cell lines to generate a cell-based functional GPCR assay. This assay employs the luminescent photoprotein aequorin and the promiscuous G-protein subunit Galpha16 and is broadly applicable to human GPCRs. We demonstrate that the assay can quantitate ligand concentration-activity relationships for seven different human GPCRs, can differentiate between partial and full agonists, and can determine rank order potencies for both agonists and antagonists that match those seen with other assay systems. Human Galpha16 improves signal strength but is not required for activity with some receptors. The coexpression of human and bovine betagamma subunits and/or phospholipase Cbeta makes no difference to agonist efficacy or potency. Two different receptors expressed in the same cell line respond to their specific agonists, and two different cell lines (Sf9 and High 5) are able to functionally detect the same expressed GPCR. Sf9 cells have the capability to produce fully functional human receptors, allied to a low background of endogenous receptors, and so are a valuable system for investigating orphan GPCRs and receptor dimerization.

The white gene encodes an ABC-type transmembrane transporter that has a role in normal eye pigment deposition. In addition, overexpression in Drosophila leads to homosexual male courtship. Its human homologue has been implicated in cholesterol transport in macrophages and in mood disorders in human males. The garnet gene is a member of a group of other Drosophila eye colour genes that have been shown, or proposed, to function in intracellular protein transport. Recent molecular analysis indicates that it encodes the δ subunit of the AP-3 adaptin complex involved in vesicle transport from the trans-Golgi network to lysosomes and related organelles, such as pigment granules. This identification revealed a novel role for intracellular vesicular transport in Drosophila pigmentation. To further analyze this intracellular transport system, we examined the genetic interactions between garnet and a second site enhancer mutation, enhancer of garnet (e(g)). We show here that e(g) is a cryptic allele of the white gene. The white-garnet interaction is highly sensitive to the levels of both gene products but also shows some allele specificity for the white gene. The additive effect on pigmentation and the predicted protein products of these genes suggest that the garnet/AP-3 transport system ensures the correct intracellular localization of the white gene product. This model is further supported by the observation of homosexual male courtship behavior in garnet mutants, similar to that seen in flies overexpressing, and presumably mis-sorting, the white gene product. The we(g)allele also enhances mutations in the subset of other eye-color genes with phenotypes similar to garnet. This observation supports a role for these genes in intracellular transport and leads to a model whereby incorrect sorting of the white gene product can explain the pigmentation phenotypes of an entire group of eye-color genes.Key words: white, garnet, e(g), AP-3, adaptins.

The garnet gene was one of the first genes to be identified in Drosophila melanogaster. Mutations in the garnet gene affect both of the biochemically distinct types of pigments in the eye and disrupt pigmentation of other organs. As an initial step in the analysis of this gene, we have analyzed the pigmentation defects in several of the garnet alleles. We have also cloned the gene and examined its expression in various tissues and at different stages of development. The garnet gene is expressed throughout development and in all tissues examined. Structurally related sequences can be detected in a variety of other eukaryotes. The predicted protein sequence of the garnet product resembles clathrin and nonclathrin adaptin proteins and is highly similar to the delta subunit of the newly isolated mammalian AP-3 adaptin complex, which is associated with the trans-Golgi network and endosomes. This suggests that garnet encodes a protein that acts in the intracellular sorting and trafficking of vesicles from the trans-Golgi network to endosomes, and related specialized organelles such as the pigment granule. This finding provides an explanation for the phenotype of garnet mutations and predicts that other Drosophila eye-colour genes will be a rich resource for the genetic dissection of intracellular vesicle transport.Key words: garnet, Drosophila melanogaster, AP-3, eye pigments.

This study presents a detailed analysis of the acidic N-terminal region of theOrgyia pseudotsugatamulticapsid nucleopolyhedrovirus (OpMNPV) transactivator IE1. The N-terminal region of IE1 is rich in acidic amino acids and has been hypothesized to be an acidic activation domain. Removal of the N-terminal 126 amino acids containing the acidic domain of IE1 resulted in complete loss of transactivation activity, indicating that this region is essential for transactivation. The OpMNPV acidic domain was replaced with the archetype acidic activation domain from VP16 and the acid-rich region ofAutographa californicamulticapsid NPV (AcMNPV) IE1. These chimeric constructs were fully capable of transactivation in transient assays. The chimeric OpMNPV IE1s containing the herpes simplex virus VP16 and AcMNPV IE1 acidic activation domains consistently transactivated a reporter gene to higher levels than the OpMNPV IE1 acidic activation domain. Transactivation by the chimeric constructs is enhanced synergistically when cotransfected with IE2 intoLymantria disparandSpodoptera frugiperdacells. Both N- to C-terminal and C- to N-terminal deletions of the OpMNPV acidic activation domain were constructed to define functional domains within the OpMNPV IE1 acidic activation domain. At least two potential activation domains were identified. Within each of these domains, two core regions at amino acids 28–43 and amino acids 113–124 were identified that were similar to core regions of VP16 and GAL4, which contain predominately acidic and bulky hydrophobic amino acids.

Suppressors of position-effect variegation (Su(var)s) in Drosophila melanogaster are usually studied in the presence of chromosomal rearrangements, which exhibit variegated expression of euchromatic genes moved near to, or heterochromatic genes moved away from, centromeric heterochromatin. However, the effects of Su(var) mutations on heterochromatic gene expression in the absence of a variegating re-arrangement have not yet been defined. Here we present a number of results which suggest that Su(var) gene products can interact to affect the expression of the light gene in its normal heterochromatic location. We initially observed that eye pigment was reduced in several Su(var) double mutants; the phenotype resembled that of light mutations and was more severe when only one copy of the light gene was present. This reduced pigmentation could be alleviated by a duplication for the light gene or by a reduction in the amount of cellular heterochromatin. In addition, the viability of most Su(var) double mutant combinations tested was greatly reduced in a genetic background of reduced light gene dosage, when extra heterochromatin is present. We conclude that Su(var) gene products can affect expression of the heterochromatic light gene in the absence of any chromosomal rearrangements. However, it is noteworthy that mutations in any single Su(var) gene have little effect on light expression; we observe instead that different pairings of Su(var) mutations are required to show an effect on light expression. Interestingly, we have obtained evidence that at least two of the second chromosome Su(var) mutations are gain-of-function lesions, which also suggests that there may be different modes of interaction among these genes. It may therefore be possible to use this more sensitive assay of Su(var) effects on heterochromatic genes to infer functional relationships among the products of the 50 or more known Su(var) loci.Key words: heterochromatin, chromatin, gene interactions.

The discovery of egg masses of Asian gypsy moth onboard Russian freighters in Pacific ports of North America and the capture of adult male gypsy moths in pheromone traps underscored the need for a positive identification of Asian and European gypsy moth males. We have devised a method for differentiation of these two populations based on restriction endonuclease cleavage of a polymerase chain reaction (PCR) generated product. DNA primers for the 5S and 28S rDNA were employed to amplify the ITS2 region using PCR. The ends of the amplified DNA products were sequenced and gypsy moth specific oligonucleotide primers were designed to amplify the gypsy moth ITS2 region. Eleven of 28 restriction enzymes tested cleaved the amplified product. The restriction enzymes ClaI, PvuI, and TaqI generated distinct restriction fragment patterns for the Asian and European gypsy moth PCR product. This diagnostic was applied to samples of field-collected gypsy moths in a double blind test and correctly identified them as Asian or European. Additional studies demonstrated that these primers and restriction enzymes could be used to differentiate among Lymantria dispar, L. monacha, and L. mathura.

Transposable elements may be potential tools for the dispersal of engineered DNA through target insect populations. The utility of this hypothesis is predicated on the ability of transposable elements carrying a large DNA insert to rapidly disperse through a population. In addition, the inserted DNA must be replicated with a high degree of fidelity during this dispersal. We have monitored the ability of a transposable element with an inserted gene to spread through experimental populations and tested whether the passenger gene retains its ability to encode an active protein. Several Drosophila melanogaster laboratory populations were initiated with female flies that were null for alcohol dehydrogenase activity and contained no P elements. Most of the females were mated to males of the same strain; however, 1 or 10% of the females were mated to males from a strain that had previously been transformed with a helper P element and a P element/Adh gene construct. The dispersal of P elements to new genomes was monitored at each generation by randomly selecting females and performing DNA hybridization assays on dissected ovarian tissue. In addition, each female was tested for alcohol dehydrogenase activity using a simple histochemical assay. We find that, despite an approximate threefold increase in size, the P element constructs containing a functioning gene are still capable of rapid dispersal through the experimental populations. We also show that many of the inserted Adh genes still encode an active product.Key words: P element, transformation, Adh, transposable element.

Cytogenetic region 31 of the second chromosome of Drosophila melanogaster was screened for recessive lethal mutations. One hundred and thirty nine new recessive lethal alleles were isolated that fail to complement Df(2L)J2 (31A-32A). These new alleles, combined with preexisting mutations in the region, define 52 complementation groups, 35 of which have not previously been described. Among the new mutations were alleles of the cdc2 and mfs(2)31 genes. Six new deficiencies were also isolated and characterized identifying 16 deficiency subintervals within region 31. The new deficiencies were used to further localize three loci believed to encode non-histone chromosomal proteins. Suvar(2)1/Su(var)214, a dominant suppressor of position-effect variegation (PEV), maps to 31A-B, while the recessive suppressors of PEV mfs(2)31 and wdl were localized to regions 31E and 31F-32A, respectively. In addition, the cytological position of several mutations that interact with heterochromatin were more precisely defined.

The sites of seven tRNA genes (Arg-2, Lys-2, Ser-2b, Ser-7, Thr-3, Thr-4, Val-3b) were studied by in situ hybridization.125I-labeled tRNA probes fromDrosophila melanogaster were hybridized to spreads of polytene chromosomes prepared from fourDrosophila species representing different evolutionary lineages (D. melanogaster, Drosophila hydei, Drosophila pseudoobscura, andDrosophila virilis). Most tRNA loci occurred on homologous chromosomal elements of all four species. In some cases the number of hybridization sites within an element varied and sites on nonhomologous elements were found. It was observed that both tRNA 2 Arg and tRNA 2 Lys hybridized to the same site on homologous elements in several species. These data suggest a limited amount of exchange among different linkage groups during the evolution ofDrosophila species.

The mus209B1 mutant of Drosophila melanogaster exhibits a complex pleiotropy of temperature-sensitive (ts) lethality, hypersensitivity to DNA-damaging agents such as ionizing radiation and methyl methanesulfonate, suppression of position-effect variegation (PEV), and female sterility. Our discovery that mus209 encodes proliferating cell nuclear antigen (PCNA), which is an indispensable component of the DNA replication apparatus, suggests that alterations to chromosome replication may underlie that pleiotropy. Nine lethal mutations, three of them ts, genetically define the Pcna locus. Temperature shift studies reveal that the vital function of PCNA is required throughout virtually all stages of fly development, and that maternally encoded PCNA is essential for embryogenesis. All three ts mutants strongly suppress PEV, which suggests a role for PCNA in chromatin assembly or modification.

In fission yeast, the product of the cdc2 gene is required both for entry into S phase and mitosis. Homologs of cdc2 have been isolated from a number of metazoans, but in general they have not been amenable to genetic analysis. Here we describe P element transposon tagging of Cdc2 in Drosophila melanogaster and the analysis of 10 Cdc2 mutants. The recessive lethality of Cdc2P is associated with a P element located in the 5′ untranslated region of the gene. Seven other alleles have unique single base pair substitutions in the coding region of Cdc2. One allele, Cdc2B47, is mutated in the splice donor site of exon 1. Most mutations in Cdc2, including the presumptive null allele Cdc2B47, die at the pupal stage, suggesting that the maternally supplied Cdc2 gene product drives earlier cell divisions. The phenotypes of our mutants are consistent with a role for Cdc2 in cell proliferation; however, we did not observe any perturbation of the endoreduplication cycle associated with the acquisition of polyteny.Key words: Cdc2, Drosophila, mutations, sequence.

Characterization of a group of dominant second chromosome suppressor of position-effect variegation (PEV) (Su(var)) mutants has revealed a variety of interesting properties, including: maternal-effect suppression of PEV, homozygous lethality or semilethality and male-specific hemizygous lethality, female infecundity, acute sensitivity to the amount of heterochromatin in the cell and sensitivity to sodium butyrate. Deficiency/duplication mapping and complementation tests have revealed that eight of the mutants define at least two genes in section 31 of the left arm of chromosome 2 and they suggest that a ninth corresponds to an additional nonessential Su(var) gene within or near this region. The effects of specific deficiencies and a duplication on PEV indicate that the expression of one or more of the Su(var) genes in this region of the chromosome is dose-dependent, i.e., capable of haplo-abnormal suppression and triplo-abnormal enhancement. Interestingly, the appearance of certain visible phenotypes among a subset of the mutants suggests that they may possess antimorphic properties. Our results are consistent with the hypothesis that two of these Su(var) genes encode structural components of heterochromatin. We also report that two previously isolated mutants located in 31E and 31F-32A act as recessive suppressors of PEV.

Variant tRNA genes have been widely observed in multicellular eukaryotes. Recent biochemical studies have shown that some of them are expressed in a tissue- or a stage-specific manner. These findings would thus imply that certain modified tRNAs may be crucial for the development of the organism. Using Drosophila melanogaster as a model, we have taken a combined genetic and molecular approach to examine critically the possible biological functions of tRNA(4, 7Ser) genes. We showed that at least 50% of the total templates can be deleted from the genome without inducing abnormal phenotypes such as Minute, or a decrease in viability. In addition, two of the tRNASer variant genes that are unique in sequence are also completely dispensable. This strongly implies that even though they may be expressed in vivo, they play no essential role in the development of the fruitfly. By comparison with some of the corresponding tRNA genes in another sibling species, Drosophila erecta, our results suggest strongly that the variants are products non-reciprocal exchanges among the tRNA(4, 7Ser), genes. Such intergenic recombination events may have a major influence in the concerted evolution of the two gene families.

We examined the genetic, morphological, and molecular effects of position effect variegation in Drosophila, and the effects of mutations that either suppress [Su(var)] or enhance [E(var)] this phenomenon. All eight Su(var) mutations examined strongly suppress the inactivation of variegating alleles of the genes white [In(l) wm4], brown [In(2R) bwVDe2] and Stubble [T(2; 3) SbV]. The E(var) mutation enhances variegation of these loci. The chromosomal region 3C-E (26 bands) which includes the white locus is usually packaged as heterochromatin in salivary glands of the variegating strain wm4. Addition of any of the Su(var) mutations restores a more euchromatic morphology to this region. In situ hybridization to polytene chromosomes and DNA blot analyses of gene copy number demonstrate that the DNA of the w+ gene is less accessible to its probe in the variegating wm4 strain than it is in the wild-type or variegation-suppressed strains. Blot analysis of larval salivary gland DNA indicates that the white gene copy number does not vary among the strains. Hence, the differences in binding of the w+ gene probe in the variegating and variegation-suppressed strains reflect differences in chromosomal packaging rather than alterations in gene number. The effects of variegation and the Su(var) mutations on chromatin structure were analyzed further by DNAse I digestion and DNA blot hybridization. In contrast to their dramatic effects on chromosomal morphology and gene expression, the Su(var) mutations had negligible effects on nuclease sensitivity of the white gene chromatin. We suggest that the changes in gene expression resulting from position effect variegation and the action of the Su(var) mutations involve alterations in chromosomal packaging.

Six purified tRNAs labeled with 125I by chemical or enzymatic methods were hybridized to polytene chromosomes of Drosophila melanogaster. The main chromosomal regions of hybridization wer: tRNAGly(GGA), 58A, 84C, and 90E; tRNALeu(2), 44E, 66B5-8, and 79F; tRNASer(2b), 86A, 88A9-12, and 94A6-8; tRNAThr(3), 47F and 87B; tRNAThr(4), 93A1-2; and tRNATyr(1 gamma), 19F, 22F-23A, 41, 50C1-4 and 85A. At 50C the hybridization of tRNATyr(1 gamma) was polymorphic in the giant strains. When the hybridization of three valine isoacceptors studied previously was re-investigated, it was found that only one hybridization site, 90BC, was shared between tRNAVal(3b) and tRNAVal(4). tRNAVal(3a) did not have any sites in common with the other two.

The genes for tRNA3bval were localized to 84D and 92B on the polytene chromosomes of Drosophila melanogaster with a possible minor site at 90B-C by hybridization in situ and autoradiography with 125I-labeled tRNA3bval. Flies carrying a duplication of the 84D region had increased amounts (30%) of tRNA3bval in proportion to the increased number of genes. While a proportional decrease in the amount of tRNAval3b in flies bearing a deletion of the same region was found, the total acceptance of valine remained at the level found in the wild type.

We have undertaken the study of a collection of 32 Drosophila melanogaster mus strains selected on the basis of developmental sensitivity to the DNA-damaging agents, methyl methanesulfonate (MMS), N-acetyl-2-aminofluorene (AAF), nitrogen mustard (HN2), and gamma-radiation. In total, 18 of these strains are sensitive to MMS. In turn, 14 of these exhibit unconditional MMS sensitivity (one of the latter mutants is lethal at 29 degrees C), whereas the other 4 are sensitive to MMS only at higher temperatures. Detailed analysis of the 7 strongest MMS-sensitive strains reveals that they identify 4 new second chromosome mus loci. Two mus loci are each represented by two alleles. One mutant (mus205B1) is allelic to a previously characterized mus locus. Different MMS-sensitive mutants display patterns of mutagen cross-sensitivity (to AAF, HN2, benzo[a]pyrene (BP), and gamma-rays) that parallel the range of responses seen in previously recovered X-linked and autosomal mus loci. In general, mutations that are strongly sensitive to MMS are also sensitive to one or both of the procarcinogens, AAF and BP, as opposed to HN2 and gamma-radiation. In contrast, the moderately MMS-sensitive mutations are sensitive to HN2 and gamma-rays, but not to AAF or BP. Of the 14 mus strains that are not sensitive to MMS, 5 are sensitive to AAF, another 5 are sensitive to HN2, and the remaining 4 are sensitive to gamma-rays.

Highly purified tRNAs from Drosophila melanogaster were iodinated with 125I and hybridized to squashes of polytene chromosomes of Drosophila silivary glands followed by autoradiography to localize binding sites. Most tRNAs hybridize strongly to more than one site and weakly to one or more additional sites. The major sites for various tRNAs are the following: tRNA2Arg, 42A, 84F1,2; tRNA2Asp, 29DE; tRNA3Gly, 22BC, 35BC, 57BC, tRNA2Lys, 42A, 42E; tRNA5Lys, 84AB, 87B; tRNA2Met, 48B5-7, 72F1-2, 83F-84A; tRNA3Met, 46A1-2, 61D1-2, 70F1-2; tRNA4Ser, 12DE, 23E; tRNA7Ser, 12DE, 23E; tRNA3aVal, 64D; tRNA3bVal, 84d3-4, 92b1-9; tRNA4Val, 56D3-7, 70BC.

Essentially nothing is known of the origin, mode of transmission, and evolution of mobile elements within the genus Drosophila. To better understand the evolutionary history of these mobile elements, we examined the distribution and conservation of homologues to the P, I, gypsy, copia, and F elements in 34 Drosophila species from three subgenera. Probes specific for each element were prepared from D. melanogaster and hybridized to genomic DNA. Filters were washed under conditions of increasing stringency to estimate the similarity between D. melanogaster sequences and their homologues in other species. The I element homologues show the most limited distribution of all elements tested, being restricted to the melanogaster species group. The P elements are found in many members of the subgenus Sophophora but, with the notable exception of D. nasuta, are not found in the other two subgenera. Copia-, gypsy-, and F-element homologues are widespread in the genus, but their similarity to the D. melanogaster probe differs markedly between species. The distribution of copia and P elements and the conservation of the gypsy and P elements is inconsistent with a model that postulates a single ancient origin for each type of element followed by mating-dependent transmission. The data can be explained by horizontal transmission of mobile elements between reproductively isolated species.

We have previously reported that four tRNAs of Drosophila melanogaster randomly labeled with iodine-125 hybridize in part to the 56EF region of polytene chromosomes where 5S RNA genes occur. In the presence of a 100-fold excess of unlabeled 5S RNA no hybridization of randomly labeled 125I-tRNA Asp2 gamma occurred at 56EF although hybridization elsewhere was not affected. In addition, tRNAAsp2 gamma labeled by introducing 125I-5-iodocytidylyl residues into the 3'-CCA end with tRNA nucleotidyl transferase did not hybridize to 56EF but did hybridize to other sites. The hybridization of tRNALys2, tRNA3Gly and tRNAMet3 at 56EF was not eliminated by a 25 to 100-fold excess of unlabeled 5S RNA. When these tRNAs were labeled at the -CCA terminus they hybridized to 56EF as well as to their other sites with the exception that terminally labeled tRNALys2 no longer hybridized to 62A. The hybridization of the latter three species of tRNA to the region of the 5S genes, amongst other sites, is confirmed. The previously observed hybridization of tRNAAsp2 gamma in this region appears to have been due to contamination of the tRNA sample with traces of material derived from 5S RNA.