Multiple mating and sperm displacement in a natural population of Drosophila melanogaster

Summary Mother-offspring data for alcohol dehydrogenase genotypes of a vineyard cellar population of D. melanogaster are best explained by a model that allows 21% of females in the population to mate twice with an 83% level of sperm displacement. A population model with multiple mating and sperm displacement is examined theoretically. A formula for the effective population size is derived under this model. Multiple mating increases the effective population size relative to single mating.     

Surface markers of a purified peritoneal eosinophil population from Mesocestoides corti-infected BALB/c male mice

Eosinophils of approximately 95% purity were prepared from the peritoneal cavities of BALB/c male mice infected with larval cestode, Mesocestoides corti. The alloantigenic surface marker phenotype of this cell population was shown to be H-2+Ly4+Ly5+Lyt-1-,2-,3-Ly-6-,7-Ia-Thy-1-TL-. Two of four anti-Lyt-2 sera were positive when tested on purified eosinophils by using the Staphylococcus aureus protein A sheep erythrocyte rosetting method, but absorption studies indicated that this reaction was not due to anti-Lyt-2 antibodies. Eosinophils are therefore Lyt-2-, although some Lyt-2 sera contain additional eosinophil reactive antibodies. A proportion (20 to 40%) of the population of eosinophils was positive for the Fc receptor, but all were negative for the C3 receptor and for surface immunoglobulin.     

Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition,bi-directionally from the primed protospacer

Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼350 new spacers acquired in priming events and identified a 5′-protospacer-GG-3′ protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2–3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.     

Development of a bifunctional crosslinking agent with potential for the preparation of immunotoxins

A new protein crosslinking agent, 2,3-dibromopropionyl-N-hydroxysuccinimide ester, has been synthesized and characterized. The potential use of this compound as a temperature-controllable heterobifunctional crosslinking agent has been investigated using model systems and its reactivity compared with that of chlorambucil-N-hydroxysuccinimide ester. The coupling of¹⁴C-labeled phenylethylamine to lysozyme has been used to illustrate the feasibility of the use of this crosslinking agent for the synthesis of immunotoxins.     

Gene transfection of the HuLy-m2 (Leu-9) antigen into mouse L cells

Human DNA was transfected into mouse L cells and tk⁺ HuLy-m2⁺ (= CD7⁺) transfectants isolated after growth in hypoxanthine, aminopterin, thymidine medium and repeated cloning. After several cycles of transfection, > 90% of HuLy-m2⁺ L cells could be detected, by rosetting and by cytofluorography, which showed the transfectants to have a density of CD7 two to five times that found on peripheral blood lymphocytes. Despite this, the 37 kd CD7⁺ dimer could only be identified with difficulty using cell-surface radioiodination and sodium dodecyl sulfate-polyacrylamide gel electrophoresis techniques. An antiserum was produced (C3H anti-HuLy-m2⁺ L cells) which, after absorption, was shown to react with HuLy-m2 antigens present on human thymocytes and lymphocytes and on CD7⁺ transfected L cells.Abbreviations BSA bovine serum albumin - DME Dulbecco's modified Eagle's medium - EDTA ethylenediamine-tetraacetate - HAT hypoxanthine, aminopterin, thymidine - HSV herpes simplex virus - PBL peripheral blood lymphocyte - PBS phosphate-buffered saline - RFC rosette-forming cell - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - tk thymidine kinase     

The mouse Fc receptor for IgG (Ly-17) : molecular cloning and specificity

A cDNA clone encoding the mouse Ly-17⁺ Fc receptor for IgG, isolated from a myelomonocytic cell line, was sequenced and expression of mRNA and the functional FcR investigated. The receptor is a 301 amino acid transmembrane glycoprotein with two homologous extracellular domains that are also homologous to members of the Ig superfamily. The receptor has four sites of N-linked glycosylation and a long 94 amino acid cytoplasmic tail. Northern analysis, immune complex binding, and serological studies demonstrate that the receptor encoded by the cDNA clone binds mouse IgG_1/2b and rabbit IgG complexes.     

Effects of viral chemotherapeutic agents on membrane properties. Studies of cyclosporin A, benzyloxycarbonyl-D-Phe-L-Phe-Gly and amantadine

Cyclosporin A, benzyloxycarbonyl-D-Phe-L-Phe-Gly, and amantadine inhibit the dilution of fluorescently labeled lipids, as measured with the resonance energy exchange assay for membrane fusion. The fusion was studied using sonicated vesicles containing 1,2-dioleoyl-sn-glycero(3)phosphoethanolamine, egg (3-sn-phosphatidyl)choline, and cholesterol in a 1:1:1.3 molar ratio. All three antiviral agents inhibited myelin basic protein-induced membrane fusion when present at low concentrations in the membrane. The mechanism by which these agents affect membrane properties was investigated. The effect of these agents on the bilayer to hexagonal phase transition of 1,2-dielaidoyl-sn-glycero(3)phosphoethanolamine was determined using both differential scanning calorimetry and 31P NMR. Benzyloxycarbonyl-D-Phe-L-Phe-Gly is particularly effective in raising the bilayer to hexagonal phase transition temperature while cyclosporin promotes the greatest amount of broadening of the 31P NMR signal. Both effects are suggested to be related to the inhibitory activity of these substances on membrane fusion and possibly also to their antiviral activity.     

Antibodies which recognize the C-terminus of the inhibitory guanine-nucleotide-binding protein (Gi) demonstrate that opioid peptides and foetal-calf serum stimulate the high-affinity GTPase activity of two separate pertussis-toxin substrates.

We investigated the mechanisms of receptor-mediated stimulation of high-affinity GTPase activity in response to opioid peptides and to foetal-calf serum in membranes of the neuroblastoma X glioma hybrid cell line NG108-15. Increases in GTPase activity in response to both of these ligands was abolished by prior exposure of the cells to pertussis toxin. Pertussis toxin in the presence of [32P]NAD+ catalysed incorporation of radioactivity into a broad band of approx. 40 kDa in membranes prepared from untreated, but not from pertussis-toxin-pretreated, cells. Additivity studies indicated that the responses to opioid peptides and to foetal-calf serum were mediated by separate guanine-nucleotide-binding proteins (G-proteins). Whereas opioid peptides produced an inhibition of adenylate cyclase in membranes of untreated cells, foetal-calf serum did not. Affinity-purified antibodies which recognize the C-terminus of the inhibitory G-protein identified a 40 kDa polypeptide in membranes of NG108-15 cells. These antibodies attenuated opioid-stimulated high-affinity GTPase activity, but did not markedly affect the response to foetal-calf serum. We conclude that receptors for the opioid peptides function via the inhibitory G-protein (Gi), whereas foetal-calf serum activates a second pertussis-toxin-sensitive G-protein, which has a C-terminal sequence significantly different from that of Gi.