The effect of distamycin A on heterochromatin condensation of Drosophila chromosomes

Larval neuroblasts of four species of Drosophila (melanogaster, hydei, virilis and funebris) were treated with distamycin A, a DNA ligand which induces distinct undercondensation in AT-rich heterochromatin. For each species the patterns of undercondensation were correlated with distribution of quinacrine-bright regions and of satellite DNAs. An overlapping of distamycin A-sensitive and quinacrine-bright heterochromatic regions was demonstrated for D. melanogaster and D. virilis, but not for D. hydei and D. funebris. Distamycin A undercondensation is thus a further criterion for resolving heterochromatin into different parts and enables identification of the steps of the condensation process within heterochromatic regions.     

Comparison of dot chromosome sequences from D. melanogaster and D. virilis reveals an enrichment of DNA transposon sequences in heterochromatic domains

Background

Chromosome four of Drosophila melanogaster, known as the dot chromosome, is largely heterochromatic, as shown by immunofluorescent staining with antibodies to heterochromatin protein 1 (HP1) and histone H3K9me. In contrast, the absence of HP1 and H3K9me from the dot chromosome in D. virilis suggests that this region is euchromatic. D. virilis diverged from D. melanogaster 40 to 60 million years ago.

Results

Here we describe finished sequencing and analysis of 11 fosmids hybridizing to the dot chromosome of D. virilis (372,650 base-pairs) and seven fosmids from major euchromatic chromosome arms (273,110 base-pairs). Most genes from the dot chromosome of D. melanogaster remain on the dot chromosome in D. virilis, but many inversions have occurred. The dot chromosomes of both species are similar to the major chromosome arms in gene density and coding density, but the dot chromosome genes of both species have larger introns. The D. virilis dot chromosome fosmids have a high repeat density (22.8%), similar to homologous regions of D. melanogaster (26.5%). There are, however, major differences in the representation of repetitive elements. Remnants of DNA transposons make up only 6.3% of the D. virilis dot chromosome fosmids, but 18.4% of the homologous regions from D. melanogaster; DINE-1 and 1360 elements are particularly enriched in D. melanogaster. Euchromatic domains on the major chromosomes in both species have very few DNA transposons (less than 0.4 %).

Conclusion

Combining these results with recent findings about RNAi, we suggest that specific repetitive elements, as well as density, play a role in determining higher-order chromatin packaging.     

Purification and characterization of diaphorases from someDrosophila species

Diaphorase-1 and diaphorase-2 were isolated from twoDrosophila species,D. virilis andD. melanogaster, and purified by gel filtration, affinity chromatography, immunoaffinity chromatography, and ion-exchange chromatography. The molecular weights of both enzymes were the same in each species. The molecular weight of diaphorase-1 was the same under both denaturating and nondenaturating conditions, close to 60,000, indicating a monomeric structure. Sodium dodecyl sulfate (SDS) electrophoresis of the purified diaphorase-2 revealed the presence of a single protein band of 55,000 Da, while the molecular weight of the native enzyme was found to be 67,000. The two diaphorases were further characterized by their pH optima, isoelectric points, and kinetic parameters, and antibodies were raised in rabbits against the purified enzymes fromD. virilis. The antibodies showed no cross-reactions but recognized the corresponding diaphorases inD. melanogaster andD. novamexicana as well asD. virilis. The data obtained confirmed the hypothesis of an independent genetic control of diaphorase-1 and diaphorase-2 inDrosophila.     

Protein metabolism of Drosophila male accessory glands—II: Species-specificity of secretion proteins

The male accessory gland proteins of six Drosophila species belonging to three subgenera are compared with respect to their one- and two-dimensional electrophoretic properties. The species are: D. melanogaster, D. busckii, D. funebris, D. hydei, D. nigromelanica and D. virilis. In addition, the enhancing effect of the gland's secretion on ovulation is examined in heterospecific secretion injection experiments. Clear-cut differences in the electrophoretic protein patterns of the six species are observed on native and dissociative gels. The patterns reveal that the species differ with regard to the occurrence of some protein fractions and the relative abundance of others. The heterospecific secretions fail to induce ovulation in D. melanogaster. The results thus provide unequivocal evidence that the male accessory gland products in Drosophila are highly species-specific.     

The insect SNMP gene family

SNMPs are membrane proteins observed to associate with chemosensory neurons in insects; in Drosophila melanogaster, SNMP1 has been shown to be essential for the detection of the pheromone cis-vaccenyl acetate (CVA). SNMPs are one of three insect gene clades related to the human fatty acid transporter CD36. We previously characterized the CD36 gene family in 4 insect Orders that effectively cover the Holometabola, or some 80% of known insect species and the 300 million years of evolution since this lineage emerged: Lepidoptera (e.g. Bombyx mori, Antheraea polyphemus, Manduca sexta, Heliothis virescens, Helicoverpa assulta, Helicoverpa armigera, Mamestra brassicae); Diptera (D. melanogaster, Drosophila pseudoobscura, Aedes aegypti, Anopheles gambiae, Culex pipiens quinquefasciatus); Hymenoptera (Apis mellifera); and Coleoptera (Tribolium castaneum). This previous study suggested a complex topography within the SNMP clade including a strongly supported SNMP1 sub-clade plus additional SNMP genes. To further resolve the SNMP clade here, we used cDNA sequences of SNMP1 and SNMP2 from various Lepidoptera species, D. melanogaster and Ae. aegypti, as well as BAC derived genomic sequences from Ae. aegypti as models for proposing corrected sequences of orthologues in the D. pseudoobscura and An. gambiae genomes, and for identifying orthologues in the B. mori and C. pipiens q. genomes. We then used these sequences to analyze the SNMP clade of the insect CD36 gene family, supporting the existence of two well supported sub-clades, SNMP1 and SNMP2, throughout the dipteran and lepidopteran lineages, and plausibly throughout the Holometabola and across a broad evolutionary time scale. We present indirect evidence based on evolutionary selection (dN/dS) that the dipteran SNMPs are expressed as functional proteins. We observed expansions of the SNMP1 sub-clade in C. pipiens q. and T. castaneum suggesting that the SNMP1s may have an expanded functional role in these species.     

Surface structure of the compound eye of various Drosophila species and eye mutants of Drosophila melanogaster

Summary The surface structure of the compound eyes of 6 Drosophila species and 12 eye mutants of D. melanogaster were compared by scanning electron microscopy. D. melanogaster, D. simulans, D. hydei, D. funebris and D. virilis displayed hexagonal facets and differed only slightly in the distribution of bristles. D. lebanonensis displayed tetragonal facets.No obvious differences in surface structure of the eyes of colour mutants of D. melanogaster were found. Mutants with structural modifications of the eyes revealed irregular patterns of bristles, variations in bristle number and variations in facet shape, size and organization. The mutant spa^pol does not display clear-cut delineated facets.     

Effects of adenine on the stability and expression of xanthine dehydrogenase from Drosophila virilis

The properties of xanthine dehydrogenase from Drosophila virilis have been studied and the effects of adenine on the enzyme analysed. The enzyme has a much higher thermal stability than that from D. melanogaster. Ammonium sulphate fractionation of the extracts precipitated the enzyme in the 0–40% saturation stage whereas the D. melanogaster enzyme precipitates between 40–60% saturation. Xanthine dehydrogenase activity per mg protein in crude extracts is 40–45% lower in D. virilis than in D. melanogaster but it is more resistant to dialysis. In vitro addition of adenine inhibits both enzymes but does not appear to stabilise the D. virilis enzyme during dialysis.     

Nascent glycoproteins with a chitin-like carbohydrate component in Drosophila melanogaster imaginal discs

The incorporation of [1-³H]d-glucosamine in Drosophila melanogaster imaginal discs revealed the synthesis of glycoproteins represented by a family of subfractions with roughly the same molecular mass of about 80,000 and discrete isoelectric point values in the range of 5.0 to 6.5 pH units. The incorporation of [1-³H]d-glucosamine was not inhibited by tunicamycin, an inhibitor of N-glycosylation. This family of glycoproteins is relatively protease-resistant but can be digested by high concentrations of pronase E (100 μg/ml) or pepsin (1 mg/ml). The carbohydrate component of these glycoproteins is sensitive to chitinase. The properties of the glycoproteins in imaginal discs are similar to those of chitinase sensitive glycoproteins found in established cell cultures of D. melanogaster [Kramerov et al., Insect Biochem. 16, 417–432 (1986)]. Incorporation of [1-³H]d-glucosamine into the family of glycoproteins decreases as the imaginal discs undergo evagination induced by 20-hydroxyecdysone.     

Aurora,a non-mobile retrotransposon in Drosophila melanogaster heterochromatin

A novel retrotransposon, aurora, containing 324 by long terminal repeats (LTRs) was detected in Drosophila melanogaster as a 5 kb insertion in the heterochromatic Stellate gene. This insertion causes a 5 bp duplication of the integration site. Southern analysis and in situ hybridization data show that all detectable copies of aurora are immobilized in the D. melanogaster heterochromatin. However, mobile copies of aurora were revealed in the cuchromatin of D. simulans. The element was also found in various species of the melanogaster subgroup and in the D. virilis genome.     

Electron microscope heteroduplex study of Drosophila mitochondrial DNAs: evolution of A+T-rich region.

Mitochondrial DNAs (mtDNA) from three species of the genus Drosophila (D. melanogaster, D. simulons, and D. virilis) were compared by electron microscope heteroduplex mapping. Analysis of heteroduplex molecules revealed that the A + T-rich region of these mtDNAs has undergone quite extensive base sequence divergence, whereas the remainder of the molecule was found to share apparently complete base sequence homology in all three species. The differences in the sizes of the A + T-rich regions, as determined from the heteroduplex measurements, completely account for the differences in the total sizes of these mtDNAs. A segment of approximately 0.1 kb is conserved within the A + T-rich regions of D. simulans and D. virilis mtDNAs, but not within the A + T-rich region of D. melanogaster mtDNA. HaeIII restriction endonuclease analysis of the heteroduplex molecules has further shown that the unique HaeIII site of D. virilis mtDNA molecule is apparently conserved in both D. melanogaster and D. simulans mtDNA molecules. Finally, electrophoretic patterns of HaeIII-digested mtDNAs from all three species were found to be different and distinguishable from each other suggesting that single base substitutions have probably taken place throughout the entire mitochondrial genome.     

Small heat shock proteins and adaptation of various Drosophila species to hyperthermia

The dynamics and the level of accumulation of small heat shock proteins (sHSP group 21–27) after a heat exposure were studied in three Drosophila species differing in thermotolerance. The southern species Drosophila virilis, having the highest thermotolerance, surpassed thermosensitive D. lummei and D. melanogaster in the level of sHSPs throughout the temperature range tested. The results suggest an important role of sHSPs in the molecular mechanisms of adaptation to adverse environmental conditions, particularly to hyperthermia.     

Structural and functional analysis of a new retrotransposon class in Drosophila species

Several copies of the Penelope transposable element, previously described in Drosophila virilis, have been studied in different D. virilis strains and D. melanogaster strains transformed with P-based constructs bearing a full-size Penelope copy. Most Penelope copies in both species have large terminal inverted repeats (TIRs) and deletions of various sizes at the 5′ ends of their ORFs. Junctions between TIRs and ORFs usually have microhomologies of various lengths, which allowed a hypothesis explaining the emergence of these complex structures at the molecular level to be put forward. Most Penelope copies have a 34 bp long direct repeat at the ORF ends. Full-size and truncated Penelope copies are usually surrounded by target site duplications of various lengths.     

Effects of inhibitors and activators on the activity of two diaphorases from Drosophila virilis

The effect of some inhibitors and activators of mammalian DT-diaphorase on diaphorase-1 (DIA-1) and diaphorase-2′ (DIA-2′) purified from Drosophila virilis was studied. The inhibitors and activators changed the activity of these diaphorases in a different way, revealing a similarity between mammalian DT-diaphorase and D. virilis DIA-1 on the one hand and on the other between the D. virilis DIA-1 and the diaphorase purified from Bombyx mori eggs. These effects also confirm the independent genetic control of DIA-1 and DIA-2′ in D. virilis and make possible the differentiation of these diaphorase activities in crude enzyme extracts.     

Adaptation to a Starch Environment and Regulation of alpha-Amylase in Drosophila

The adaptation to glucose and starch foods insix species, D. melanogaster, D.virilis, D. saltans, D. funebris,D. levanonensis and D. americana, wasstudied by measuring productivity. D.melanogaster and D. virilis adapted more to thestarch environment than to the glucose environment,while D. saltans adapted more to the glucoseenvironment than to the starch environment. D.funebris, D. levanonensis, and D. americana did not distinctlyadapt to either environment. In addition, the regulationof amylase in the six species was investigated bymeasuring the levels of amylase activity with glucoseand starch food environments. The levels of amylaseactivity in D. levanonensis and D.saltans were substantially low, indicating thatthese species cannot utilize starch as a carbon source.The starch-adapted species, D. melanogaster and D.virilis, showed higher levels of amylase activitywith the starch environment and higher inducibility.These results suggest that changing the regulation ofamylase is important for the adaptation to a starch environment inDrosophila.     

The hobo transposable element has transposase-dependent and-independent excision activity in drosophilid species

Mobility of the hobo transposable element was determined for several strains of Drosophila melanogaster and several Drosophila species. Mobility was assessed by use of an in vivo transient assay in the soma of developing embryos, which monitored hobo excision from injected indicator plasmids. Excision was detected in a D. melanogaster strain (cn; ry ⁴²) devoid of endogenous hobo elements only after co-injection of a helper plasmid containing functional hobo transposase under either heat shock or normal promoter regulation. Excision was also detected in D. melanogaster without helper in strains known to contain genomic copies of hobo. In Drosophila species confirmed not to contain hobo, hobo excision occurred at significant rates both in the presence and absence of co-injected helper plasmid. In four of the seven species tested, excision frequencies were two- to fivefold lower in the presence of plasmid-borne hobo. hobo excision donor sites were sequenced in indicator plasmids extracted from D. melanogaster cn; ry ⁴² and D. virilis embryos. In the presence of hobo transposase, the predominant excision sites were identical in both species, having breakpoints at the hobo termini with an inverted duplication of proximal insertion site DNA. However, in the absence of hobo transposase in D. virilis, excision breakpoints were apparently random and occurred distal to the hobo termini. The data indicate that hobo is capable of functioning in the soma during embryogenesis, and that its mobility is unrestricted in drosophilids. Furthermore, drosophilids not containing hobo are able to mobilize hobo, presumably by a hobo-related cross-mobilizing system. The cross-mobilizing system in D. virilis is not functionally identical to hobo with respect to excision sequence specificity.     

Heat-shock DNA homology in distantly related species of Drosophila

Polytene chromosomes of D. melanogaster and D. virilis were hybridized in situ with ¹²⁵I labeled mRNA isolated from polysomes of D. melanogaster tissue culture cells incubated at 37° C. ¹²⁵I mRNA hybridized preferentially with subdivisions 87A and 87Cl of the D. melanogaster 3R chromosome; grains were also observed at regions 93D, 95D and over the chromocenter. A considerable cross hybridization of this mRNA with D. virilis polytene chromosomes was observed. The 29C region of the D. virilis second chromosome was the main site of hybridization. Significant grain numbers also appeared in region 20F of the same chromosome. The two regions mentioned belong to heat shock loci in the latter species. Based on label intensity we conclude that region 29C of D. virilis contains DNA sequences retaining molecular homology with those at subdivisions 87A and 87Cl of D. melanogaster. SDS-polyacrylamide gel electrophoresis revealed similar distributions of heat shock proteins in the two species studied.     

Oviposition pattern of several Drosophila species under various light environments

The oviposition pattern over two continuous days under constant light (LL) and light-and-dark (LD) conditions was studied by means of a special apparatus; four Drosophila species (D. lutescens, D. melanogaster, D. hydei, and D. virilis) were used. The results showed that: (1) Under the LL condition, the oviposition pattern was characteristic for each species. The number of eggs laid by D. lutescens was smaller than that laid by the other species, and the oviposition pattern of this species was rhythmical. Females of D. melanogaster laid eggs continuously, but at a low rate. In contrast, D. hydei and D. virilis laid eggs in clusters, the total numbers of eggs being larger than those of D. lutescens and D. melanogaster. (2) Females of three species (D. lutescens, D. melanogaster, and D. virilis) laid more eggs in the light phase than in the dark phase under the LD condition. However, no consistent trend was obtained with D. hydei, suggesting that the oviposition pattern of this species is indifferent to the light condition (i.e., it is independent of it). (3) No effects of pre-experimental light condition on the oviposition were found for any of the species. The light-dependency of the oviposition pattern is discussed from the viewpoint of adaptation.     

Synthesis of novel multivalent fluorescent inhibitors with high affinity to prostate cancer and their biological evaluation

Prostate-specific membrane antigen (PSMA) is an important biological target for therapy and diagnosis of prostate cancer. In this study, novel multivalent PSMA inhibitors with glutamate-urea-lysine structures were designed to improve inhibition characteristics. Precursors of the novel inhibitors were prepared from glutamic acid with di-tert-butyl ester. A near-infrared molecular dye, sulfo-Cy5.5, was introduced into the precursors to generate the final PSMA fluorescent inhibitors, compounds 12–14, to visualize prostate cancer. Biological behaviors of the inhibitors were evaluated using in vitro inhibition assays, in vivo fluorescent imaging, and ex vivo biodistribution assays. K_i values from inhibition studies indicated that dimeric inhibitor 13 with a glutamine linker showed approximately 3-fold more inhibitory activity than monomeric inhibitor 12. According to other biological studies using a mouse model of prostate cancer, dimeric inhibitor compounds 13 and 14 had higher tumor accumulation than the monomer. However, glutamine-based dimeric inhibitor 13 showed lower liver uptake than dimeric inhibitor 14, which had a benzene structure. Thus, these studies suggest that glutamine-based dimeric inhibitor 13 can be a promising optical inhibitor of prostate cancer.     

Evolutionary Changes in Gene Expression,Coding Sequence and Copy-Number at the Cyp6g1 Locus Contribute to Resistance to Multiple Insecticides in Drosophila

Widespread use of insecticides has led to insecticide resistance in many populations of insects. In some populations, resistance has evolved to multiple pesticides. In Drosophila melanogaster, resistance to multiple classes of insecticide is due to the overexpression of a single cytochrome P450 gene, Cyp6g1. Overexpression of Cyp6g1 appears to have evolved in parallel in Drosophila simulans, a sibling species of D. melanogaster, where it is also associated with insecticide resistance. However, it is not known whether the ability of the CYP6G1 enzyme to provide resistance to multiple insecticides evolved recently in D. melanogaster or if this function is present in all Drosophila species. Here we show that duplication of the Cyp6g1 gene occurred at least four times during the evolution of different Drosophila species, and the ability of CYP6G1 to confer resistance to multiple insecticides exists in D. melanogaster and D. simulans but not in Drosophila willistoni or Drosophila virilis. In D. virilis, which has multiple copies of Cyp6g1, one copy confers resistance to DDT and another to nitenpyram, suggesting that the divergence of protein sequence between copies subsequent to the duplication affected the activity of the enzyme. All orthologs tested conferred resistance to one or more insecticides, suggesting that CYP6G1 had the capacity to provide resistance to anthropogenic chemicals before they existed. Finally, we show that expression of Cyp6g1 in the Malpighian tubules, which contributes to DDT resistance in D. melanogaster, is specific to the D. melanogaster–D. simulans lineage. Our results suggest that a combination of gene duplication, regulatory changes and protein coding changes has taken place at the Cyp6g1 locus during evolution and this locus may play a role in providing resistance to different environmental toxins in different Drosophila species.