Studies on the Sex-Specific Lethals of DROSOPHILA MELANOGASTER. IV. Gynandromorph Analysis of Three Male-Specific Lethals, mle, msl-227 and mle(3)132

Mutants at three male-specific lethal loci of Drosophila melanogaster (mle, msl-2²⁷ and mle(3)132) were examined by gynandromorph analysis. In all cases only a very few gynandromorphs with small X/O patches appeared. Most of these small X/O patches were in the abdomen, and the structures in these X/O regions were reduced in size. These results indicate that the primary effects of these mutants are not on any particular organs or tissues, but rather on individual cells. mle and msl-2 have been shown by Belote and Lucchesi (1980a) to be defective in dosage compensation in X/Y males. We suggest that this dosage-compensation defect results in the expression of Minute-like phenotypes in X/O cells, and hence results in the death of X/O males and flies with large X/O tissue areas.     

Changes in the levels of prostaglandins and thromboxane and their roles in the accumulation of exudate in rat carrageenin-induced pleurisy — a profile analysis using gas chromatography-mass spectrometry —

Injection of γ-carrageenin into t he pleural cavity of rats caused the accumulation of the pleural exudate. When levels of prostaglandins (PGs) and thromboxane (TX) B₂ were quantified by gas chromatography-mass spectrometry as their methyl ester (ME)-dimethyllisopropylsilyl (DMiPS) ether or ME-methoxine-DMiPS ether derivatives, 6-keto-PGF_1α reached the maximum at 1 hr after carrageenin, then PGE₂ and TXB₂ showed peaks at 3 hr and waned off before 9 hr. he PGF_2α level was kept low, but PGD₂, PGE₁ and PGF_1α were not detected. Aspirin (100 mg/kg, i.p.) significantly decreased the PG and TXB₂ levels and suppressed the rate of plasma exudation until 5 hr, but did not at 7 hr, when it was measured by the amount of exuded pontamine sky blue injected intravenously. OKY-025 (300 mg/kg, i.p.), a selective TXA synthetase inhibitor, and tranylcypromine (20 mg/kg, i.p.), a PGI synthetase inhibitor, could not extensively inhibit the accumulation of the exudate. These results suggest that the cyclooxygenase products of arachidonic acid, particularly PGE₂, definitely play an important role in the exudation during the first 5 hr.     

Chromosome segregation in an asymmetrically dividing bacterium,Caulobacter crescentus

The mode of chromosome segregation in an asymmetrically dividing bacterium, Caulobacter crescentus, was studied by examining the fate of labeled DNA strands. Swarmer cells (one type of Caulobacter daughter cell), in which single strands of DNA had been labeled with [³H]thymidine during the previous round of chromosome replication, were grown synchronously in a non-radioactive medium for two generations. The distribution of radioactivity among the cells was visualized by autoradiography under a phase-contrast microscope. The labeled DNA strands in each cell were found to consist of two conserved units. From this, we propose a model in which the swarmer cell has two identical chromosomes, which are segregated into the progeny swarmer cell and the progeny stalked cell after chromosome replication.     

Deletion mapping and heterogenote analysis of a mutation responsible for osmosis-sensitive growth, spectinomycin resistance, and alteration of cytoplasmic membrane in Escherichia coli

Lambda transducing phages carrying Escherichia coli deoxyribonucleic acid of various lengths from the aroE-rpsL region were lysogenized into the F'3 plasmid and were used for heterogenote analysis of YM101, a sucrose-dependent, spectinomycin-resistant mutant of E. coli. Three characteristics of the mutant strain, resistance to spectinomycin, sucrose dependence of growth, and lack of I-19 protein in the cytoplasmic membrane, were shown to be the result of a mutation in a region designated delta 53-spcl. This region extends over 3.6-kilobase pairs and is located within a cluster of ribosomal genes. The mutation is recessive to the wild-type allele.     

Scanning electron-microscopy of starch granules, with or without amylase attack

Scanning electron-microscopy (SEM) revealed that, for starch granules relatively susceptible to amylase, numerous pin holes could be observed on the surfaces of granules attacked by amylase. We also observed that the pores penetrated into the inner layers of granules during the enzyme action and some of the granules exhibited a terraced or step-shaped apperance in their inner portions. These internal characteristics are most probably indicative of layered internal structures of the granules. The other characteristic observations by SEM were striated structures on the surfaces of starch granules of banana, lily, and lotus attacked by pancreatin.     

Degradation of rat cardiac myofibrils and myofibrillar proteins by a myosin-cleaving protease.

The degradation of rat cardiac myofibrils and their constituent proteins with a myosin-cleaving protease was studied. Electrophoretograms of the digestion products of myofibrils showed that myosin,M-protein, C-protein, and troponin were degraded, but actin and tropomyosin were not. Degradation of these constituents resulted in losses of the Mg2+-ATPase activity and its Ca2+-sensitivity of myofibrils. Incubation of myofibrils with the protease induced the release of alpha-actinin without degradation. Susceptibilities of myosin, actin, troponin, and alpha-actinin purified from rat and pig hearts to the protease were essentially identical to those of the assembled forms in myofibrils. Although the purified tropomyosin was readily degraded into five fragments with the protease, the tropomyosin assembled in myofibrils and actin-tropomyosin complex were insusceptible to the protease. Digestion of myosin in the filamentous state with the protease resulted in the disappearance of myosin heavy chain and light chain 2, producing two fragments having molecular weights of 130,000 and 94,000 which originated from the degradation of heavy chain. The Ca2+- and EDTA-ATPase activities of the degradation products remained unchanged during incubation for 22 h. The actin-activated ATPase activity of myosin was reduced by 30% during incubation for 6 h, and recovered to the original level on adding actin to give a ratio of actin to myosin of 2:1. The pH optima for degradation of myosin in the soluble and filamentous states were 8.5 and 7.0, respectively. The results indicate that cardiac myosin in the filamentous state was more readily degraded with the protease than the myosin in the soluble state.     

Reaction of fungal amine oxidase with beta-bromoethylamine.

beta-Br-ethylamine is both a substrate and an irreversible inhibitor of amine oxidase from Aspergillus niger. The enzyme catalyzes the nonoxidative elimination of HBr from beta-Br-ethylamine to form acetaldehyde. beta-Br-ethylamine meets several criteria for an irreversible substrate analog or suicide inhibitor. 1) It inactivates the oxidized enzyme, but not the reduced enzyme. 2) The Michaelis constant for beta-Br-ethylamine in the elimination reaction showed a similar magnitude to that of the related constant found when the haloamine acted as an inhibitor. 3) The enzyme was protected from the inactivation by the co-existence of the substrate. 4) Inactivation with beta-Br-[14C]ethylamine resulted in the incorporation of radioactivity corresponding to 1 mol of the label/mol of the monomeric unit of the enzyme and a decrease of 1 mol of the -SH group. 5) Inactivation was accompanied by the formation of a new absorption peak at 320 nm which was bleached by addition of NaBH4.     

Antibodies introduced into living cells by red cell ghosts are functionally stable in the cytoplasm of the cells.

The function and fate of antibodies introduced into living cells by red cell ghosts were studied using CRM 176 (a mutant diphtheria toxin having lower toxicity than the wild-type) and antibody against fragment A of diphtheria toxin. IgG labeled with iodine and FITC was found in the cytoplasm of the recipient cells. When about 1500 molecules of anti-fragment A antibody (rabbit IgG) were introduced into diphtheria toxin-sensitive Vero cells or FL cells, these cells became resistant to the toxin and formed normal colonies. It was calculated from the survival of cells without anti-fragment A IgG under these conditions that about 300 molecules of fragment A-176 were transferred to the cells. These results showed that the antigen-antibody reaction took place in living cells as effectively as in a cell-free system. The functional stability of antibody IgG in cells was examined by exposing Vero cells containing a subminimal amount of anti-fragment A IgG (about 1000 molecules) to the toxin for 2 hr at various times after the introduction of anti-fragment A IgG. More than 50% of the initial activity of the antibody to neutralize toxin still remained even after incubation of the cells at 37°C for 20 hr. The same degree of stability was also demonstrated using iodine-labeled specific anti-fragment A IgG. The IgG recovered from the recipient cells after various times of incubation at 37°C retained its full ability to bind to fragment A-conjugated Sepharose 4B, although the total amount of IgG associated with the cells decreased about 50% in 24 hr.     

Continuous production of glucose-6-phosphate by immobilized Achromobacter butyri cells

Summary Whole cells of Achromobacter butyri OUT 8004 having polyphosphate glucokinase activity were immobilized in polyacrylamide gel. The immobilized cells were activated by organic solvents, especially acetone. The immobilization resulted in increased stability of polyphosphate glucokinase. Continuous high yield production of G-6-P from glucose and metaphosphate was performed with an immobilized cell column, which had a half-life of approximately 20 days.Abbreviations G-6-P glucose-6-phosphate - G-1-P glucose-1-phosphate - Cation-S stearyl trimethyl ammonium chloride - SDS sodium dodecyl sulfate - Tris tris(hydroxymethyl)-aminomethane; p-NPP, p-nitrophenyl phosphate - S.V. space velocity