Multiple paternity in two natural populations (orchard and vineyard) of Drosophila.

Male mating success is an important fitness component in Drosophila. The seminal fluid conveyed with the sperm inhibits the proclivity of the female to remate and reduces her fitness. Nevertheless, females may remate before they have exhausted the sperm from the first male and consequently use sperm from both males. We have studied concurrent multiple paternity (CMP) in two Drosophila melanogaster populations, from an apple orchard and a vineyard just after harvest. CMP is high in both populations, somewhat greater than 50%; but it is not significantly higher in the vineyard, where the population density is much greater than in the orchard. Population density had been thought to be an important determinant of CMP incidence. We have used four gene loci coding for enzymes as independent markers for detecting CMP.

The Drosophila p70s6k homolog exhibits conserved regulatory elements and rapamycin sensitivity.

The protein p70s6k/p85s6k lies on a mitogen-stimulated signaling pathway and plays a key role in G1 progression of the cell cycle. Activation of this enzyme is mediated by a complex set of phosphorylation events, which has largely contributed to the difficulty in identifying the upstream kinases that mediate p70s6k activation. Genetics has proved a powerful complementary approach for such problems, providing an alternative means to identify components of signaling cascades and their functional end targets. As a first step toward implementing such an approach, we have cloned cDNAs encoding the Drosophila melanogaster p70s6k homolog (Dp70s6k). Dp70s6k is encoded by a single gene, which generates three mRNA transcripts and exhibits an overall identity of 78% in the catalytic domain with its mammalian counterpart. Importantly, this high identity extends beyond the catalytic domain to the N terminus, linker region, and the autoinhibitory domain. Furthermore, all the critical phosphorylation sites required for mammalian p70s6k activation are conserved within these same domains of Dp70s6k. Chief amongst these conserved sites are those associated with the selective rapamycin-induced p70s6k dephosphorylation and inactivation. Consistent with this observation, analysis of total S6 kinase activity in fractionated Drosophila Schneider line 2 cell extracts reveals two peaks of activity, only one of which is rapamycin sensitive. By employing a monospecific polyclonal antibody generated against Dp70s6k, we show that the cloned DP70s6k cDNA has identity with only the rapamycin sensitive peak, suggesting that this biological system would be useful in determining not only the mechanism of p70s6k activation, but also in elucidating the mechanism by which rapamycin acts to inhibit cell growth.

Mutations in the small subunit of the Drosophila U2AF splicing factor cause lethality and developmental defects.

The essential eukaryotic pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein auxiliary factor) is required to specify the 3' splice at an early step in spliceosome assembly. U2AF binds site-specifically to the intron polypyrimidine tract and recruits U2 small nuclear ribonucleoprotein to the branch site. Human U2AF (hU2AF) is a heterodimer composed of a large (hU2AF65) and small (hU2AF35) subunit. Although these proteins associate in a tight complex, the biochemical requirement for U2AF activity can be satisfied solely by the large subunit. The requirement for the small subunit in splicing has remained enigmatic. No biochemical activity has been found for hU2AF35 and it has been implicated in splicing only indirectly by its interaction with known splicing factors. In the absence of a biochemical assay, we have taken a genetic approach to investigate the function of the small subunit in the fruit fly Drosophila melanogaster. A cDNA clone encoding the small subunit of Drosophila U2AF (dU2AF38) has been isolated and sequenced. The dU2AF38 protein is highly homologous to hU2AF35 containing a conserved central arginine- and serine-rich (RS) domain. A recessive P-element insertion mutation affecting dU2AF38 causes a reduction in viability and fertility and morphological bristle defects. Consistent with a general role in splicing, a null allele of dU2AF38 is fully penetrant recessive lethal, like null alleles of the Drosophila U2AF large subunit.

Expression of Drosophila mushroom body mutations in alternative genetic backgrounds: a case study of the mushroom body miniature gene (mbm).

Mutations in 12 genes regulating Drosophila melanogaster mushroom body (MB) development were each studied in two genetic backgrounds. In all cases, brain structure was qualitatively or quantitatively different after replacement of the "original" genetic background with that of the Canton Special wild-type strain. The mushroom body miniature gene (mbm) was investigated in detail. mbm supports the maintenance of MB Kenyon cell fibers in third instar larvae and their regrowth during metamorphosis. Adult mbm1 mutant females are lacking many or most Kenyon cell fibers and are impaired in MB-mediated associative odor learning. We show here that structural defects in mbm1 are apparent only in combination with an X-linked, dosage-dependent modifier (or modifiers). In the Canton Special genetic background, the mbm1 anatomical phenotype is suppressed, and MBs develop to a normal size. However, the olfactory learning phenotype is not fully restored, suggesting that submicroscopic defects persist in the MBs. Mutant mbm1 flies with full-sized MBs have normal retention but show a specific acquisition deficit that cannot be attributed to reductions in odor avoidance, shock reactivity, or locomotor behavior. We propose that polymorphic gene interactions (in addition to ontogenetic factors) determine MB size and, concomitantly, the ability to recognize and learn odors.

Molecular characterization of the mosquito vitellogenin receptor reveals unexpected high homology to the Drosophila yolk protein receptor.

The mosquito (Aedes aegypti) vitellogenin receptor (AaVgR) is a large membrane-bound protein (214 kDa when linearized) that mediates internalization of vitellogenin, the major yolk-protein precursor, by oocytes during egg development. We have cloned and sequenced two cDNA fragments encompassing the entire coding region of AaVgR mRNA, to our knowledge the first insect VgR sequence to be reported. The 7.3-kb AaVgR mRNA is present only in female germ-line cells and is abundant in previtellogenic oocytes, suggesting that the AaVgR gene is expressed early in oocyte differentiation. The deduced amino acid sequence predicts a 202.7-kDa protein before posttranslational processing. The AaVgR is a member of the low density lipoprotein receptor superfamily, sharing significant homology with the chicken (Gallus gallus) VgR and particularly the Drosophila melanogaster yolk protein receptor, in spite of a very different ligand for the latter. Distance-based phylogenetic analyses suggest that the insect VgR/yolk protein receptor lineage and the vertebrate VgR/low density lipoprotein receptor lineage diverged before the bifurcation of nematode and deuterostome lines.

Naturally occurring variation in copia expression is due to both element (cis) and host (trans) regulatory variation.

Significant differences in levels of copia [Drosophila long terminal repeat (LTR) retrotransposon] expression exist among six species representing the Drosophila melanogaster species complex (D. melanogaster, Drosophila mauritiana, Drosophila simulans, Drosophila sechellia, Drosophila yakuba, and Drosophila erecta) and a more distantly related species (Drosophila willistoni). These differences in expression are correlated with major size variation mapping to putative regulatory regions of the copia 5' LTR and adjacent untranslated leader region (ULR). Sequence analysis indicates that these size variants were derived from a series of regional duplication events. The ability of the copia LTR-ULR size variants to drive expression of a bacterial chloramphenicol acetyltransferase reporter gene was tested in each of the seven species. The results indicate that both element-encoded (cis) and host-genome-encoded (trans) genetic differences are responsible for the variability in copia expression within and between Drosophila species. This finding indicates that models purporting to explain the dynamics and distribution of retrotransposons in natural populations must consider the potential impact of both element-encoded and host-genome-encoded regulatory variation to be valid. We propose that interelement selection among retrotransposons may provide a molecular drive mechanism for the evolution of eukaryotic enhancers which can be subsequently distributed throughout the genome by retrotransposition.

Evolutionary changes in the expression pattern of a developmentally essential gene in three Drosophila species.

The hypothesis that morphological evolution may largely result from changes in gene regulation rather than gene structure has been difficult to test. Morphological differences among insects are often apparent in the cuticle structures produced. The dopa decarboxylase (Ddc) and alpha-methyldopa hypersensitive (amd) genes arose from an ancient gene duplication. In Drosophila, they have evolved nonoverlapping functions, including the production of distinct types of cuticle, and for Ddc, the production of the neurotransmitters, dopamine and serotonin. The amd gene is particularly active in the production of specialized flexible cuticles in the developing embryo. We have compared the pattern of amd expression in three Drosophila species. Several regions of expression conserved in all three species but, surprisingly, a unique domain of expression is found in Drosophila simulans that does occur in the closely related (2-5 million years) Drosophila melanogaster or in the more remote (40-50 million years) Drosophila virilis. The "sudden" appearance of a completely new and robust domain of expression provides a glimpse of evolutionary variation resulting from changes in regulation of structural gene expression.

CHD1 is concentrated in interbands and puffed regions of Drosophila polytene chromosomes.

Previously, we reported on the discovery and characterization of a mammalian chromatin-associated protein, CHD1 (chromo-ATPase/helicase-DNA-binding domain), with features that led us to suspect that it might have an important role in the modification of chromatin structure. We now report on the characterization of the Drosophila melanogaster CHD1 homologue (dCHD1) and its localization on polytene chromosomes. A set of overlapping cDNAs encodes an 1883-aa open reading frame that is 50% identical and 68% similar to the mouse CHD1 sequence, including conservation of the three signature domains for which the protein was named. When the chromo and ATPase/helicase domain sequences in various CHD1 homologues were compared with the corresponding sequences in other proteins, certain distinctive features of the CHD1 chromo and ATPase/helicase domains were revealed. The dCHD1 gene was mapped to position 23C-24A on chromosome 2L. Western blot analyses with antibodies raised against a dCHD1 fusion protein specifically recognized an approximately 210-kDa protein in nuclear extracts from Drosophila embryos and cultured cells. Most interestingly, these antibodies revealed that dCHD1 localizes to sites of extended chromatin (interbands) and regions associated with high transcriptional activity (puffs) on polytene chromosomes from salivary glands of third instar larvae. These observations strongly support the idea that CHD1 functions to alter chromatin structure in a way that facilitates gene expression.

Mutations in the rotated abdomen locus affect muscle development and reveal an intrinsic asymmetry in Drosophila.

In bilateral animals, the left and right sides of the body usually present asymmetric structures, the genetic bases of whose generation are still largely unknown [CIBA Foundation (1991) Biological Asymmetry and Handedness, CIBA Foundation Symposium 162 (Wiley, New York), pp. 1-327]. In Drosophila melanogaster, mutations in the rotated abdomen (rt) locus cause a clockwise helical rotation of the body. Even null alleles are viable but exhibit defects in embryonic muscle development, rotation of the whole larval body, and helical staggering of cuticular patterns in abdominal segments of the adult. rotated abdomen is expressed in the embryonic mesoderm and midgut but not in the ectoderm; it encodes a putative integral membrane glycoprotein (homologous to key yeast mannosyltransferases). Mesodermal cells defective in O-glycosylation lead to an impaired larval muscular system. We propose that the staggering of the adult abdominal segments would be a consequence of the relaxation of intrinsic rotational torque of muscle architecture, preventing the colateral alignment of the segmental histoblast cells during their proliferation at metamorphosis.

Age-specific inbreeding depression and components of genetic variance in relation to the evolution of senescence.

Two major theories of the evolution of senescence (mutation accumulation and antagonistic pleiotropy) make different predictions about the relationships between age, inbreeding effects, and the magnitude of genetic variance components of life-history components. We show that, under mutation accumulation, inbreeding decline and three major components of genetic variance are expected to increase with age in randomly mating populations. Under the simplest version of the antagonistic pleiotropy model, no changes in the severity of inbreeding decline, dominance variance, or the genetic variance of chromosomal homozygotes are expected, but additive genetic variance may increase with age. Age-specific survival rates and mating success were measured on virgin males, using lines extracted from a population of Drosophila melanogaster. For both traits, inbreeding decline and several components of genetic variance increase with age. The results are consistent with the mutation accumulation model, but can only be explained by antagonistic pleiotropy if there is a general tendency for an increase with age in the size of allelic effects on these life-history traits.

Translational regulation of mammalian and Drosophila citric acid cycle enzymes via iron-responsive elements.

The posttranscriptional control of iron uptake, storage, and utilization by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) provides a molecular framework for the regulation of iron homeostasis in many animals. We have identified and characterized IREs in the mRNAs for two different mitochondrial citric acid cycle enzymes. Drosophila melanogaster IRP binds to an IRE in the 5' untranslated region of the mRNA encoding the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (SDH). This interaction is developmentally regulated during Drosophila embryogenesis. In a cell-free translation system, recombinant IRP-1 imposes highly specific translational repression on a reporter mRNA bearing the SDH IRE, and the translation of SDH-Ip mRNA is iron regulated in D. melanogaster Schneider cells. In mammals, an IRE was identified in the 5' untranslated regions of mitochondrial aconitase mRNAs from two species. Recombinant IRP-1 represses aconitase synthesis with similar efficiency as ferritin IRE-controlled translation. The interaction between mammalian IRPs and the aconitase IRE is regulated by iron, nitric oxide, and oxidative stress (H2O2), indicating that these three signals can control the expression of mitochondrial aconitase mRNA. Our results identify a regulatory link between energy and iron metabolism in vertebrates and invertebrates, and suggest biological functions for the IRE/IRP regulatory system in addition to the maintenance of iron homeostasis.

Phosphorylation of the fused protein kinase in response to signaling from hedgehog.

The hedgehog gene (hh) of Drosophila melanogaster exerts both short- and long-range effects on cell patterning during development. The product of hedgehog is a secreted protein that apparently acts by triggering an intra-cellular signaling pathway, but little is known about the details of that pathway. The Drosophila gene fused (fu) encodes a serine/threonine-protein kinase that genetic experiments have implicated in signaling initiated by hedgehog. Here we report that the fused protein is phosphorylated during the course of Drosophila embryogenesis, as a result of hedgehog activity. In cell culture, phosphorylation of fused protein occurs in response to the biologically active form of hedgehog and cannot be blocked by activation of protein kinase A, which is thought to be an antagonist of signaling from hedgehog. These results suggest that fused and protein kinase A function downstream of hedgehog but in parallel pathways that eventually converge distal to fused. The reconstruction of signaling from hedgehog in cell culture should provide further access to the mechanisms by which hedgehog acts.

Ecdysone-inducible gene expression in mammalian cells and transgenic mice.

During metamorphosis of Drosophila melanogaster, a cascade of morphological changes is triggered by the steroid hormone 20-OH ecdysone via the ecdysone receptor, a member of the nuclear receptor superfamily. In this report, we have transferred insect hormone responsiveness to mammalian cells by the stable expression of a modified ecdysone receptor that regulates an optimized ecdysone responsive promoter. Inductions reaching 4 orders of magnitude have been achieved upon treatment with hormone. Transgenic mice expressing the modified ecdysone receptor can activate an integrated ecdysone responsive promoter upon administration of hormone. A comparison of tetracycline-based and ecdysone-based inducible systems reveals the ecdysone regulatory system exhibits lower basal activity and higher inducibility. Since ecdysone administration has no apparent effect on mammals, its use for regulating genes should be excellent for transient inducible expression of any gene in transgenic mice and for gene therapy.

cDNA cloning and gene mapping of a candidate human cell cycle checkpoint protein.

A family of proteins involved in cell cycle progression, DNA recombination, and the detection of DNA damage has been recently identified. One of the members of this family, human ATM, is defective in the cells of patients with ataxia telangiectasia and is involved in detection and response of cells to damaged DNA. Other members include Mei-41 (Drosophila melanogaster), Mec1p (Saccharomyces cerevisiae), and Rad3 (Schizosaccharomyces pombe), which are required for the S and G2/M checkpoints, as well as FRAP (Homo sapiens) and Torl/2p (S. cerevisiae), which are involved in a rapamycin-sensitive pathway leading to G1 cell cycle progression. We report here the cloning of a human cDNA encoding a protein with significant homology to members of this family. Three overlapping clones isolated from a Jurkat T-cell cDNA library revealed a 7.9-kb open reading frame encoding a protein that we have named FRP1 (FRAP-related protein) with 2644 amino acids and a predicted molecular mass of 301 kDa. Using fluorescence in situ hybridization and a full-length cDNA FRP1 clone, the FRP1 gene has been mapped to the chromosomal locus 3q22-q24. FRP1 is most closely related to three of the PIK-related kinase family members involved in checkpoint function--Mei-41, Mec1p, and Rad3--and as such may be the functional human counterpart of these proteins.

Spontaneous avoidance behavior in Drosophila null for calmodulin expression.

The regulatory protein calmodulin is a major mediator of calcium-induced changes in cellular activity. To analyze the roles of calmodulin in an intact animal, we have generated a calmodulin null mutation in Drosophila melanogaster. Maternal calmodulin supports calmodulin null individuals throughout embryogenesis, but they die within 2 days of hatching as first instar larvae. We have detected two pronounced behavioral abnormalities specific to the loss of calmodulin in these larvae. Swinging of the head and anterior body, which occurs in the presence of food, is three times more frequent in the null animals. More strikingly, most locomotion in calmodulin null larvae is spontaneous backward movement. This is in marked contrast to the wild-type situation where backward locomotion is seen only as a stimulus-elicited avoidance response. Our finding of spontaneous avoidance behavior has striking similarities to the enhanced avoidance responses produced by some calmodulin mutations in Paramecium. Thus our results suggest evolutionary conservation of a role for calmodulin in membrane excitability and linked behavioral responses.