Isolation and characterization of mitochondrial DNA from Drosophila melanogaster.

Mitochondrial DNA (MtDNA) with a neutral buoyant density of 1.681 g/cm(3) has been isolated from unfertilized eggs of Drosophila melanogaster. This DNA is a circular molecule with an average length of 5.3 microm; it reassociates with a low C(0)t(1/2) after denaturation, and in alkaline isopycnic centrifugation it separates into strands differing in density by 0.005 g/cm(3). MtDNA isolated from purified mitochondria of unfertilized eggs or from total larval DNA melts with three distinct thermal transitions. The three melting temperature values suggest that the molecule may have three regions differing in average base composition. DNA isolated from unfertilized eggs of D. melanogaster contains approximately equal amounts of MtDNA and another DNA with a buoyant density of 1.697 g/cm(3), slightly less dense than main peak DNA. The possibility that the heavier DNA fraction consists of amplified ribosomal DNA was excluded by hybridization experiments, but otherwise nothing is known of its origin or function.

Cellular and secretory proteins of the salivary glands of Sciara coprophila during the larval-pupal transformation.

The cellular and secretory proteins of the salivary gland of Sciara coprophila during the stages of the larval-pupal transformation were examined by electrophoresis in 0.6 mm sheets of polyacrylamide gel with both SDS-continuous and discontinuous buffer systems. After SDS-electrophoresis, all electrophoretograms of both reduced and nonreduced proteins from single glands stained with Coomassie brilliant blue revealed a pattern containing the same 25 bands during the stages of the larval-pupal transformation. With the staining procedures used in this study, qualitative increases and decreases were detected in existing proteins and enzymes. There was no evidence, however, for the appearance of new protein species that could be correlated with the onset of either pupation or gland histolysis. Electrophoretograms of reduced samples of anterior versus posterior gland parts indicated that no protein in the basic pattern of 25 bands was unique to either the anterior or posterior gland part. Electrophoretograms of reduced samples of secretion collected from either actively feeding or "cocoon"-building animals showed an electrophoretic pattern containing up to six of the 25 protein fractions detected in salivary gland samples, with varied amounts of these same six proteins in electrophoretograms of secretion samples from a given stage. Zymograms of non-specific esterases in salivary gland samples revealed a progressive increase in the amount of esterase reaction produce in one major band and some decrease in the second major band during later stages of the larval-pupal transformation.

Evidence for origin of insect sex pheromones: presence in food plants.

Compounds identified as sex attractant pheromones in a number of phytophagous insects were found in a variety of host plants. These agents vary in chemical composition in different plant species, which suggests that dietary factors may provide an evolutionary mechanism for diversification of certain insect species. A theoretical framework to explain this phenomenon is postulated on the basis of experiments with the oak leaf roller moth.

Structure, antigenic determinants of some clinically important insect allergens: chironomid hemoglobins.

Determination of the molecular structure and properties of allergens that elicit severe immediate-type hypersensitivity diseases in humans and a knowledge of the structure of their antibody-binding sites should provide new insight into the pathogenetic mechanisms of allergic diseases. Monomeric and homodimeric hemoglobins (CTT I to X) have been identified as potent allergenic components of Chironomidae, a family of Diptera. Immunologic investigations of peptides of three of these hemoglobins (CTT IV, CTT VI, and CTT VIII) showed that human antibodies of the E and G classes recognize at least two different sites within each molecule. Individual hemoglobin peptides were aligned with homologous regions of chironomid hemoglobin CTT III, whose tertiary structure has been determined by x-ray analysis at a resolution of 1.4 angstroms. The antigenic site CTT IV(91 to 101) showed the following characteristics: (i) seven polar or hydroxylated amino acids, from a total of eleven, occupying predominantly superficial regions; (ii) the property of linkage to other molecules by hydrogen bonds or solvent clusters; and (iii) high thermal mobility factors. In contrast, peptide CTT IV(102 to 108), which does not bind human antibodies, contained no polar amino acids and had low thermal mobility factors. These results support the idea that the antigenicity of clinically relevant proteins is related to regions with a predominance of polar amino acids and with low energy barriers between different conformations, which allow high flexibility, including site-specific adaptation in antibody binding.

L-leucine: a neuroactive substance in insects.

A compound was isolated from the blood of silkworm larvae, Bombyx mori, which had been prostrated with DDT; this compound increased the spontaneous discharge in the isolated abdominal nerve cord of the American cockroach, Periplaneta americana. The compound was identified as L-leucine.

Expression of the src and abl cellular oncogenes during development of Drosophila melanogaster.

Transcription of the Drosophila homologs of the abl and src cellular oncogenes, termed Dash and Dsrc , respectively, was studied. Despite the fact that Dash and Dsrc share sequence homology, they have distinct patterns of transcription during development. The Dash transcript, 6.2 kilobases long, is found in maternal RNA stored in unfertilized eggs and in embryos up to 4 h after egg laying. In contrast, Dsrc transcripts are 3.2, 4.8, and 5.2 kilobases long. They are detected in eggs and embryos and to a lesser extent in larvae and adult flies. The relative amount of each of the three Dsrc transcripts changes during the different stages of development.

Observation of tyrosine-O-phosphate in Drosophila melanogaster larvae by 31P-NMR spectroscopy.

31P-NMR spectra of intact larvae and pupae of Drosophila melanogaster have been obtained at 109.3 MHz. A major resonance in these samples has been identified as tyrosine-O-phosphate. Its chemical shift reflects the hemolymph plasma pH. Upon disruption of the organisms (necessary for chemical analyses of tyrosine-O-phosphate), phosphatases rapidly hydrolyze this phosphate ester, generating inorganic phosphate and free tyrosine.

Serial polymers in the epidermis of Drosophila melanogaster larvae.

The paper reports the existence of peculiar polymers (e-polymers) obtained from the epidermis of Drosophila melanogaster larvae. E-polymers result from the assembly of two components held together by alkali-labile bonds. Such components can be separated by CsCl density gradients and by DEAE-cellulose chromatography after controlled alkaline hydrolysis. One of the components contains predominantly neutral sugars and a phenolic substance (S-fraction). The other contains predominantly amino acids, aminosugars and a phenolic substance. This fraction can be visualized as serial multimers of a monomer subunit. It is suggested that e-polymers are continuous tridimensional structures which might have morphogenetic significance.

Developmental changes in the pattern of larval beta-globin gene expression in Xenopus laevis. Identification of two early larval beta-globin mRNA sequences.

We have analysed beta-globin mRNA sequences in total RNA extracted from embryos and tadpoles of Xenopus laevis at different stages of development and we have identified the most abundantly transcribed beta-globin mRNA (beta T1). The entire nucleotide sequence of a cDNA clone corresponding to this mRNA is known. We have now identified the gene corresponding to this mRNA and we have determined the nucleotide sequences of its immediate 5'-flanking region. Using a DNA fragment from within the coding region of the cloned beta T1 cDNA we show, by primer extension analysis, that beta T1 mRNA is first detectable at stage 28-32 of development. This is the time at which the first presumptive erythropoietic tissue, the ventral blood island, becomes observable histologically. We show that two minor beta-globin genes, distinct from beta T1, are expressed during early stages of development, and that their expression ceases shortly after the beginning of the feeding stage. We term these two early larval genes beta E1 and beta E2. A third minor beta-globin gene is expressed during early development but, unlike beta E1 and beta E2, it is also expressed throughout subsequent larval development. We term this gene beta T2 and show that it corresponds to a gene previously termed beta LII. Finally, using a primer derived from the major adult beta-globin gene (beta 1), we have analysed the accumulation of the major adult beta-globin mRNA during larval development, and we show that this sequence does not accumulate to any significant level before metamorphosis.

Circular DNA molecules in the genus Drosophila.

The satellite DNA's from the embryos of five species of Drosophila (D. melanogaster, D. simulans, D. nasuta, D. virilis and D. hydei) have been analyzed for the presence of closed circular duplex DNA molecules, as determined by CsCl-EBr gradients. Circular DNA molecules were found in every species but D. melanogaster. Analyses of cell fractions from adult Drosophila and organ fractions from Drosophila larvae show that fractions containing mitochondria are highly enriched in these molecules.

Putative nuclear triiodothyronine receptors in tadpole liver during metamorphic climax.

It has been shown previously that the maximum binding capacity (MBC) of the putative T3 receptors in tadpole red blood cells (RBCs) is increased during development and can be stimulated by treatment with thyroid hormone (TH). The present study was performed to determine if the MBC of tadpole liver nuclei is also increased during development or after treatment with TH. Because of the relatively high levels of endogenous TH in tadpoles during climax, the use of an in vivo saturation assay employing [125I]T3 was not feasible. Thus, MBC was determined by measuring by RIA the amount of T3 bound to the liver nuclei in tadpoles pretreated with sufficient T3 to saturate the receptors. Values were then corrected for the nonsaturable fraction using data obtained in tadpoles given a large dose of T3 (10 nmol). After this dose, essentially all of the T3 in the nucleus was bound to nonsaturable sites. MBC values estimated by this method and by Scatchard analysis were comparable. In contrast to the observations in tadpole RBCs, no significant change in the MBC of liver nuclei occurred as the tadpole progressed from early prometamorphosis to metamorphic climax; in tadpoles at stages XII-XIV and XIX-XXIII, MBC values were 0.308 +/- 0.024 (+/- SE) and 0.260 +/- 0.035 pmol/mg DNA, respectively. Furthermore, treatment of tadpoles with T4 (1 nmol T4; 14 days before study), which resulted in a marked increase in receptor number in RBCs, had no effect on MBC in hepatic nuclei. The amounts of nucleus-bound endogenous T3 in liver and RBCs were also determined. From these data and the MBC values, it was calculated that hepatic and RBC nuclear receptors were, respectively, 80% and more than 90% occupied with T3. These findings indicate that there is tissue specificity in the response of receptor MBC to TH during metamorphosis, and that most of the TH on the receptor during climax is T3.

The delivery of foreign genes into fertilized fish eggs using high-velocity microprojectiles.

Fertilized eggs of loach (Misgurnus fossilis), rainbow trout (Salmo gairdneri) and zebrafish (Brachydanio rerio) were bombarded with high-velocity tungsten microprojectiles covered with plasmid DNA containing sequences of beta-galactosidase and neomycin phosphotransferase genes. About 70% of the eggs survived the bombardment. The activity of both transferred genes was revealed in the fish developed from the bombarded eggs. Neomycin phosphotransferase gene sequences were detected by means of PCR amplification and Southern hybridization in the total DNA of zebrafish that survived after G418 treatment.

Serotonin-containing neurons in Drosophila melanogaster: development and distribution.

Antibodies made against serotonin (5HT) were used to identify the serotonin neuronal system in the developing and adult nervous system of Drosophila melanogaster. The 5HT neuronal pattern is composed of a small number of neurons, 84 in larvae and 106 in adults, distributed in clusters composed of one to five neurons in the CNS; 5HT immunoreactive (5HT-IR) neurons appear to be predominantly intrasegmental interneurons; however, intersegmental 5HT-IR fibers are observed and at least some neurons send peripheral fibers. Acquisition of 5HT immunoreactivity in the CNS occurs late in embryogenesis, by 16-18 hours, and most if not all the 5HT neurons appear to persist into adulthood. During early metamorphosis, the intensity of 5HT-IR neuropil transiently decreases. Other changes in the CNS during this period are reflected in the appearance of two new 5HT clusters and 5HT-IR neuropil in the developing optic lobes. Comparison of the 5HT-IR pattern with other transmitter systems in Drosophila as well as comparison of the 5HT-IR pattern within different insect species is presented.

Catecholamine-containing neurons in Drosophila melanogaster: distribution and development.

The development of catecholamine-containing neurons (CA neurons) in the fruit fly Drosophila melanogaster was studied. Glyoxylic-acid-induced histofluorescence and antibodies against dopamine and tyrosine hydroxylase were used to describe catecholamine distribution in the larval central nervous system (CNS). The three techniques gave rise to a similar pattern of distribution of putative CA neurons. At all developmental stages CA neurons were distributed widely throughout the CNS but represented only a small fraction of all CNS neurons. Catecholamine-containing processes were confined to the CNS. The CA neurons are first discerned at about 18 hours of embryonic development. We suggest that these larval CA neurons are maintained throughout the ontogeny of the fly and that the adult CA pattern is composed of embryonic neurons and neurons that differentiate during metamorphosis.

Occurrence of an active regulatory mechanism of protein synthesis during starvation and refeeding in Bombyx mori fat body.

The origin of the amino acids which participate in protein synthesis at the recovery from starvation have been determined in the fat body from Bombyx mori larvae. Endogeneous amino acids have been labelled with [3H] leucine and ingested ones with [14C] leucine, allowing their discrimination in the organism. 22 minutes after refeeding, proteosynthetic activity of the fat body, estimated by the polysome level, is increased 2.5 fold. Endogeneous leucine represents more than 90 p. cent of the leucine present in nascent polypeptides. Free leucine pools of the fat body and of hemolymph increase, mainly through the release of endogeneous leucine. It is therefore concluded that refeeding with amino acids induces the production of a signal or critical factor, responsible for the increase in proteosynthetic activity in the fat body.

Bombyx mori development: the context for its endocrine system.

Quantitative data on neurohormones (brain-hormones) steroïd hormones, juvenile hormones, in an insect : Bombyx mori are outlined. The variations of hormonal contents throughout larval and pupal development, in eggs and ovocytes are analyzed.

Lipids of stages in the life-cycle of the cestode Spirometra mansonoides.

The kinds and amounts of the lipids of each stage (egg, coracidium, procercoid, plerocercoid, adult) in the life-cycle of the cestode Spirometra mansonoides, and of environmental lipids, have been examined by combinations of TEAE-cellulose and silicic acid column, silica gel thin-layer and SCOT column gas-liquid chromatography. Major lipids of all stages were triacylglycerols, cholesterol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidylcholine. Minor lipids were sterol esters, fatty acids, benzoquinones, partial glycerides phosphatidic acid, sphingolipids and lysolipids. Triacylglycerols decreased and cholesterol and phospholipids, particularly diphosphatidylglycerol and phosphatidylcholine, increased during embryogenesis (egg to coracidium). Neutral and phosphoglycerides had characteristic fatty acyl group patterns irrespective of life-cycle stage, except for procercoid lipids, which were unique in their content of branched and odd-numbered forms. The patterns seen in the total lipids of the various life-cycle stages were qualitatively similar to those of the environments of those stages, but were often quantitatively dissimilar.

Detection of amplified Wuchereria bancrofti DNA in mosquitoes with a nonradioactive probe.

A technique to identify Wuchereria bancrofti larvae in mosquito vectors with an enzyme-labeled DNA probe is described. To overcome the low sensitivity of nonradioactive detection methods, analyte DNA was amplified by polymerase chain reaction (PCR). Oligonucleotide primers were used to amplify W. bancrofti-specific DNA fragments of 380 and 650 bp, respectively. Parasite DNA in mosquito extracts was isolated free of inhibitors of the PCR by hybridization to a biotinylated DNA fragment (IWb 67), which hybridizes to DNA from most filarial species, followed by absorption of the resulting DNA hybrids onto avidin-coated acrylic beads. PCR-amplified DNA was detected with a biotin-labeled W. bancrofti-specific repeat DNA (IWb 35) coupled to avidin-alkaline phosphatase and the chemiluminescent substrate, AMPPD. The DNA equivalent of less than one larva can be detected by this method in mosquito extracts. The sensitivity of detection was comparable to that of radioactive probes and the assay is suitable for field application in endemic countries.

Messenger RNA extracted from Schistosoma mansoni larval forms codes for parasite antigens when translated in vitro.

Intact messenger RNA was extracted from three stages of development of the parasite Schistosoma mansoni, cercariae, 3 h (mechanically prepared) schistosomula and adults. Some of the in vitro translation products are precipitable with immune rat and human serum showing that these sera recognize protein determinants. Whereas the patterns of the total translation products show major proteins expressed at all three developmental stages, immunoprecipitation delineates patterns unique to each stage. Serum from chronically infected patients is likely to preferentially recognize proteins expressed by the adult parasite yet precipitates more products of translation of cercarial RNA than of adult RNA, suggesting that many of the same epitopes are already coded by cercarial messenger RNA. It seems, however, that antigens appear on lower molecular weight species in the adult. The experiments described here open the way to cloning the genes of antigens from S. mansoni.

A monoclonal antibody specific for an epidermal cell antigen of Xenopus laevis: electron microscopic observations using a gold-labeling method.

A monoclonal antibody (EPI-1), raised against the supernatant of a homogenate of Xenopus laevis larvae at the tailbud stage (stage 36/37), interacts specifically with a 250 KD epidermal antigen of Xenopus. An immunocytochemical gold-labeling technique was used to investigate changes in antigen distribution during epidermal development of Xenopus laevis. Specific immunolabeling was initially detected over the endoplasmic reticulum in the outer epithelial cells of the late gastrula stage (stage 12.5). After the early neurula stage (stage 13), immunolabeling appeared over moderately electron-dense bodies (these bodies disappear after stage 29), and also over the apical cell surface and adjacent cytoplasm of all the outer epithelial cells. During metamorphosis, labeling decreased and disappeared after stage 62, as the superficial layer had peeled off. These data suggest that the antigen is useful as a marker of general differentiation in studies of epidermal development during the embryonic and larval stages of Xenopus laevis.