The genetic analysis of a large transposing element of Drosophila melanogaster

A member of Ising's family of large transposing elements (TEs) has inserted into, or very near, the crinkled (ck, 2–50) locus. This TE (TE36) carries functional alleles of both the white and roughest loci, and causes a hypomorphic mutation of ck. The TE is visible in polytene chromosomes as a two-banded insertion between 35B9 and 35C1. These bands show homology to foldback (FB) elements by in situ hybridization. All spontaneous losses of TE36 remain mutant for ck and retain sequences homologous to FB at the site of TE's insertion. TE36 carries only one functional copy of w ⁺, by the criterion that z w, TE36/ + flies are wild-type for eye color but z w; TE36/TE36 flies are zeste. This white⁺ gene is dosage compensated since w/Y; TE36/+ males have twice as much eye pigment as w/w; TE36/ + females. A form of the TE that has four polytene chromosome bands and expresses twice as much pigment as TE36 has been recovered. However, its white genes are not suppressed by zeste.     

Thermal stability of soluble mitochondrial H+-ATPase

ATPase melting has been studied by circular dichroism and differential scanning microcalorimetry. Decomposition of the -helix of H⁺-ATPase (in which about 80% of the peptide groups of the enzyme are involved) following thermal treatment is shown to proceed gradually, beginning with room temperature. Effect of nucleotides upon melting is detected in the range of 20–40 C. Above 40 C, the pattern of thermal decomposition of the three-dimensional structure of H⁺-ATPase is independent of the nature of nucleotides present. Highly stable -helical sites have been found in the enzyme molecule. Possible mechanism of formation of such sites is discussed, and the results obtained are compared with data on thermal stability of ATPase from thermophilic bacteria. Structural changes in the molecule following thermal treatment are compared with ATPase activity changes under similar experimental conditions.     

Interactions between WHITE Genes Carried by a Large Transposing Element and the ZESTE Allele in DROSOPHILA MELANOGASTER

TE146, a large transposing element of Drosophila melanogaster, carries two copies of the white and roughest genes in tandem. In consequence, z¹ w ^11E4; TE146(Z)/+ flies have a zeste (lemon-yellow) eye color. However, one in 10³ TE146 chromosomes mutates to a red-eyed form. The majority of these "spontaneous red" (SR) derivatives of TE146 have only one copy of the white gene and are, cytologically, two- to three-banded elements, rather than six-banded as their progenitor. The SR forms of TE146 are also unstable and give zeste-colored forms with a frequency of about one in 10⁴. One such "spontaneous zeste" (SZ) derivative carries duplicated white genes as an inverted, rather than a tandem, repeat. The genetic instability of this inverted repeat form of TE146 is different from that of the original tandem repeat form. In particular, the inverted repeat form frequently produces derivatives with internal rearrangements of the TE and gives a much lower frequency of SR forms. In addition, two novel features of the interaction between w⁺ alleles in a zeste background have been found. First, copies of w ⁺ can become insensitive to suppression by zeste even when paired. Second, an inversion breakpoint may disrupt the pairing between two adjacent w⁺ alleles, necessary for their suppression by zeste, without physically separating them.     

Control of basidiospore production by a nuclear gene in the fungus Coprinus congregatus

Summary The genetical control of basidiospore production by sporophores of the fungus Coprinus congregatus was studied. This species is characterized by a bipolar compatibility control, and homokaryons with complementary alleles A1 and A2 can be distinguished apart. We confirmed that the pale mushroom phenotype of the fungus is determined by a nuclear gene symbolized pal. This gene also controls a sporeless character and segregates independently of the mating-type locus. Dikaryons homoallelic for the pal ^– allele produce typical pale and sporeless sporophores, while heteroallelic (pal ⁺, pal ^–) and homoallelic (pal ⁺, pal ⁺) dikaryons produce normal or almost normal sporulating sporophores. In order to segregate homokaryons homoallelic for the pal gene (A1, pal ^–; A1, pal ⁺, A2, pal ^–; A2, pal ⁺), the following protocols were used: (a) the dikaryotization of stock homokaryons containing the pal ⁺ allele and of each mating type, A1 or A2, by dikaryotic mycelia homoallelic for the pal ^– allele; (b) the culturing of homokaryotic mycelia issuing from the germination of basidiospores from sporophores produced by dikaryotic mycelia heterokaryotic for the pal gene; (c) the culturing of mycelia grown from protoplasts obtained from dikaryons homoallelic for the pal ^– allele (D6 strain), and from homokaryons heteroallelic for the pal gene (H8), or homoallelic for pal ^#x002B;+ allele (H7). These techniques enabled us to segregate homokaryons of the four types defined above and were indispensable in the segregation of the pal ^– homoallelic homokaryons as no basidiospores were produced by typical pale mushrooms.     

Ganglioside-cholera toxin interactions: a binding and lipid monolayer study

Summary On t.l.c. plates ¹²⁵I-cholera toxin binds to a disialoganglioside tentatively identified as GD_lb with about 10 times less capacity than to ganglioside GM₁. Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit. Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM₁ and GD_1b. In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.Abbreviations GM₂ Gal-NAc14Gal(3-2NeuAc)14G1c1Cer - GM₁ Gal3Ga1-NAc14Gal(32NeuAc)14G1c11Cer - GD_1a NeuAc23Ga113Gal-NAc14Gal(32NeuAc)14G1c11Cer - GD1b Gall3Gal-NAcl4Gal(32NeuAc82NeuAc)14Glc11Cer - GT_1b NeuAc23Ga113Ga1-NAcal4Gal(3-2NeuAc82NeuAc)14G1c11Cer - dpPC 1,2-hexadecanoyl-sn-glycero-3-phosphocholine - dpPE 1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine     

Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: II. Comparison with transport models

Summary In this paper, the results of the preceding electrophysiological study of sodium-alanine cotransport in pancreatic acinar cells are compared with kinetic models. Two different types of transport mechanisms are considered. In the simultaneous mechanism the cotransporterC forms a ternary complexNCS with Na⁺ and the substrateS; coupled transport of Na⁺ andS involves a conformational transition between statesNCS andNCS with inward- and outward-facing binding sites. In the consecutive (or ping-pong) mechanism, formation of a ternary complex is not required; coupled transport occurs by an alternating sequence of association-dissociation steps and conformational transitions. It is shown that the experimentally observed alanine- and sodium-concentration dependence of transport rates is consistent with the predictions of the simultaneous model, but incompatible with the consecutive mechanism. Assuming that the association-dissociation reactions are not rate-limiting, a number of kinetic parameters of the simultaneous model can be estimated from the experimental results. The equilibrium dissociation constants of Na⁺ and alanine at the extracellular side are determined to beK _N <-64mm andK _S <-18mm. Furthermore, the ratioK _N /K _N ^S of the dissociation constants of Na⁺ from the binary (NC) and the ternary complex (NCS) at the extracellular side is estimated to be <-6. This indicates that the binding sequence of Na⁺ andS to the transporter is not ordered. The current-voltage behavior of the transporter is analyzed in terms of charge translocations associated with the single-reaction steps. The observed voltage-dependence of the half-saturation concentration of sodium is consistent with the assumption that a Na⁺ ion that migrates from the extracellular medium to the binding site has to traverse part of the transmembrane voltage.     

Glutathione-gated K+ channels of Escherichia coli carry out K+ efflux controlled by the redox state of the cell

The kinetics of K⁺ efflux across the membranes of i) wild-type Escherichia coli poisoned by the thiol reagent N-ethylmaleimide, ii) K⁺ retention mutants and iii) glutathione-deficient mutants, have revealed a common K⁺ leaky phenotype; it is characterized by a very high rate of K⁺ efflux. The results suggest that the products of kefB and kefC genes could encode two K⁺ channels, both gated by glutathione. The possible function of these K⁺ channels seems to be a K⁺ exit controlled by the redox state of the cell; indeed, it can be inferred from the effects of several oxidants and reductants that turning on and off of the K⁺ efflux mediated by the channels can be correlated with the redox state of glutathione.     

The role of β-dimethylsulphoniopropionate,glycine betaine and homarine in the osmoacclimation of Platymonas subcordiformis

The tertiary sulphonium compound, -dimethylsulphoniopropionate (DMSP) and the quaternary ammonium compounds glycine betaine and homarine are important osmotica in Platymonas subcordiformis cells. Following hypersaline stresses the compounds were accumulated after a lag period of 3 h and equilibrium concentrations were reached 6 h later. In contrast to these organic solutes, mannitol was synthesised immediately and equilibrium concentrations were reached within 90 min. Hyposaline stresses induced losses of the organic solutes from the cells. The ions K⁺, Na⁺, Cl^- and the above organic solutes can account for the osmotic balance of the cells.Abbreviations DMSP -dimethylsulphoniopropionate - _i intracellular osmolality - _o extracellular osmolality     

Oligomerization reactions of deoxyribonucleotides on montmorillonite clay: The effect of mononucleotide structure on phosphodiester bond formation

Adenine deoxynucleotides bind more strongly to Na⁺-montmorillonite than do the corresponding ribonucleotides. Thymidine nucleotides binds less strongly to Na⁺-montmorillonite than do the corresponding adenine deoxynucleotides. Oligomers of 2-dpA up to the tetramer were detected in the reaction 2-d-5-AMP with EDAC (a water-soluble carbodiimide) in the presence of Na⁺-montmorillonite. Reaction of 3-d-5-AMP with EDAC on Na⁺-montmorillonite yields 3-d-2,5-pApA while the reaction of 2-d-3-AMP yields almost exclusively 3,5-cdAMP. The reaction of 5-TMP under the same reaction conditions give 3,5-cpTpT and 3,5-pTpT while 3-TMP gives mainly 3,5-cpT. The yield of dinucleotide products (dpNpN) containing the phosphodiester bond is 1% or less when Na⁺-montmorillonite is omitted from the reaction mixture.     

Transductional mapping of nine linked chromosomal genes in Bacillus thuringiensis

Summary We have previously described a phage (63) for generalized transduction in Bacillus thuringiensis and used it for mapping of four chromosomal antibiotic resistance markers, namely nalA-rifA-strA-spcA (Landén et al. 1981). From 63 we have now isolated a host range mutant called 64 which contains 52–56 megadalton of DNA. Phage 64 was found to be a more efficient transducing vector than 63. The host range of 64 is wide, with good growth on subspecies gelechiae, kurstaki, galleriae, thuringiensis and thompsoni, restriction on some derivatives of finitimus and ostrinae and no growth on alesti, israelensis and aizawai.Using 64 and a series of new mutants of subspecies gelechiae we have no added five new genes to the antibiotic resistance group described before. The gene order found was guaB-purB-metA-novA-(purA-nalA)-rifA-strA-spcA. Linkage was also demonstrated between hisA and lysA.     

Prerequisite for His4 in deltorphin A for highδ opioid receptor selectivity

Summary Analysis of deltorphin A position 4 analogues included: backbone constrained N MeHis, spinacine (Spi), N MePhe and the tetrahydroisoquinoline-3-carboxylic acid (Tic); spatially confined side-chain (Phg); and imidazole alkylation ofl- andd-His⁴ enantiomers. High selectivity was lost with the following replacements: N MeHis⁴, N MePhe⁴ and Phg⁴ reduced binding and the constrained residues also increasedµ binding; ring closure between the side-chain and amino group to yield Spi⁴ or Tic⁴ increasedµ affinity. Imidazole methylation of His⁴ marginally affected opioid binding and doubled selectivity; alkylatedd-His⁴-derivatives generally maintained selectivity in spite of decreased affinities. Thus, His⁴ imidazole preserves selectivity by facilitating high binding and by repulsion at theµ receptor. Several low energy conformers of deltorphin A indicated that the His⁴ imidazole preferred a spatial orientation parallel to the phenolic side-chain of Tyr¹ suggestive that this conformation might contribute to high affinity and selectivity.     

The mechanism of Na+ transport by rabbit urinary bladder

Summary The mechanism of Na⁺ transport in rabbit urinary bladder has been studied by microelectrode techniques. Of the three layers of epithelium, the apical layer contains virtually all the transepithelial resistance. There is radial cell-to-cell coupling within this layer, but there is no detectable transverse coupling between layers. Cell coupling is apparently interrupted by intracellular injection of depolarizing current. The cell interiors are electrically negative to the bathing solutions, but the apical membrane of the apical layer depolarizes with increasingI _sc. Voltage scanning detects no current sinks at the cell junctions or elsewhere. The voltage-divider ratio, , (ratio of resistance of apical cell membrane,R _a, to basolateral cell membrane,R _b) decreases from 30 to 0.5 with increasingI _sc, because of the transportrelated conductance pathway in the apical membrane. Changes in effective transepithelial capacitance withI _sc are predicted and possibly observed. The transepithelial resistance,R _t, has been resolved intoR _a, R_b, and the junctional resistance,R _j, by four different methods: cable analysis, resistance of uncoupled cells, measurements of pairs of (R _t, ) values in the same bladder at different transport rates, and the relation betweenR _t andI _sc and between andI _sc.R _j proves to be effectively infinite (nominally 300 k F) and independent ofI _sc, andR _a decreases from 154 to 4 k F with increasingI _sc. In the resulting model of Na⁺ transport in tight epithelia, the apical membrane contains an amiloride-inhibited and Ca⁺⁺-inhibited conductance pathway for Na⁺ entry; the basolateral membrane contains a Na⁺–K⁺-activated ATPase that extrudes Na⁺; intracellular (Na⁺) may exert negative feedback on apical membrane conductance; and aldosterone acts to stimulate Na⁺ entry at the apical membrane via the amiloride-sensitive pathway.     

Tryptic digestion of the (Na + K)-ATPase is both sensitive to and modifies K+ interactions with the enzyme

Tryptic digestion of the (Na + K)-ATPase in the presence of choline chloride or NaCl (Na-type) and in the presence of KCl (K-type) produced distinct patterns of peptide fragments and losses of catalytic activity. TheK _0.5 for K⁺ to shift digestion from the Na-type, and its sensitivity to dimethyl sulfoxide and Triton X-100, were consistent with K⁺ acting at sites on the cytoplasmic face of the enzyme through which the K-phosphatase reaction also is activated. Reagents favoring the E₁ conformational states, oligomycin, Triton, and ATP, shifted the pattern toward the Na-type, whereas those favoring E₂ states, dimethyl sulfoxide, MgCl₂, and MnCl₂, shifted the pattern toward the K-type. Na-type digestion caused a greater loss of K-phosphatase than (Na + K)-ATPase activity, and the residual K-phosphatase activity was more sensitive to inhibition by Triton and ATP but stimulated more by dimethyl sulfoxide and inhibited less by P_i and MnCl₂; all these effects are consistent with such digestion shifting equilibria toward E₁ enzyme states. Accordingly, theK _0.5 for K⁺ to activate the (Na + K)-ATPase was increased. However, theK _0.5 for the K-phosphatase was unchanged; this observation requires revision of previous formulations, and bears on additional aspects of enzyme activity as well.     

A novel eye morphology induced by a P element in somatic tissue of Drosophila melanogaster.

Summary We found a specific eye morphology designated as Square, which is induced when some Drosophila melanogaster strains harboring P elements are crossed with the 2–3 strain carrying a modified P element, P[ry ⁺, 2–3], which produces transposase in somatic tissue. This phenotype was dominant and also induced in the reciprocal crosses. Square was induced when the 2–3 strain was crossed with Q and M strains such as the sn^w (M) strain carrying three small P elements but not with P strains. Inheritance of Square was also tested and its phenotype was not transmitted to the next generation. These results suggest that Square is caused by the transposition of P elements in somatic cells.     

Biosynthesis of RNA polymerase in Escherichia coli

Summary Effect of temperature-sensitive, assembly-defective mutations in Escherichia coli RNA polymerase (rpoB) or subunit gene (rpoC) was investigated on the expression of wild-type rpoB ⁺C⁺operon, which was introduced by infection of a lambda transducing phage drif ⁺ (rpoB ⁺)-6 after UV-irradiation of the mutant cells. In rpoB2·rpoB7 strain which accumulates assembly-intermediates, free , ₂ complex and premature core, the expression of rpoB ⁺C⁺operon measured by the rate of subunit synthesis was considerably inhibited whereas that of EF(translation elongation factor)-Tu, ribosomal proteins L1 and L7/L12, and some -coded proteins remained unaffected. On the other hand, the expression was enhanced specifically for only rpoB ⁺C⁺operon in either rpoC4 or rpoC1 mutants, which are defective in the association of ₂ complex and subunit or the activation of premature core enzyme, respectively. Upon preincubation of the mutant cells at 42° C prior to phage infection, during which assembly intermediates degraded rapidly, the rate of subunit synthesis relative to other phage-corded proteins increased remarkably in rpoB2·rpoB7 mutant as well as in rpoC4 and rpoC1 mutants. These observations strongly suggested the autogenous regulation for at least (rpoB ⁺C⁺) operon by some trans-active diffusible protein complexes built of RNA polymerase subunits. Nature of the regulatory molecules is discussed.Paper VI in this series is Saitoh and Ishihama (1977)     

Phenotypic instability in a tif-1 mutant of Escherichia coli

Summary A recent study showed that in E. coli T44 () carrying the tif-1 mutation, elevated temperature and adenine can interfere with the translation process. The present study shows that the expression of tif phenotypes (thermoinduction and filamentation) is suppressed by factors which affect ribosomal function. Ethanol suppresses thermoinduction and, in some spc ^r mutants, both thermoinduction and filamentation are suppressed. An unknown factor(s) in yeast extract suppresses both thermoinduction and filamentation. In thermoresistant revertant (ts⁺), the expression of the ts⁺ phenotype is suppressed by yeast extract, ethanol, guanosine+cytidine and by the addition of a spc ^r mutation. This indicates that this phenotype could be due to suppressor mutations, and the interaction between factors affecting ribosomal function and the ts⁺ phenotype suggests that the suppression of tif in the ts⁺ strains could operate on the ribosomal level. In vitro studies show that in extracts from either spc ^r or ts⁺ strains, or in the presence of ethanol, translational restriction is relieved, suggesting that the suppression of tif phenotypes could involve the translation process.     

Effect of chemical modifiers of amino acid residues on proton conduction by the H+-ATPase of mitochondria

The effect of chemical modifiers of amino acid residues on the proton conductivity of H⁺-ATPase in inside out submitochondrial particles has been studied. Treatment of submitochondrial particles prepared in the presence of EDTA (ESMP) with the arginine modifiers, phenylglyoxal or butanedione, or the tyrosine modifier, tetranitromethane, caused inhibition of the ATPase activity. Phenylglyoxal and tetranitromethane also caused inhibition of the anaerobic release of respiratory H⁺ in ESMP as well as in particles deprived of F₁ (USMP). Butanedione treatment caused, on the contrary, acceleration of anaerobic proton release in both particles. The inhibition of proton release caused by phenylglyoxal and tetranitromethane exhibited in USMP a sigmoidal titration curve. The same inhibitory pattern was observed with oligomycin and withN,N-dicyclohexylcarbodiimide. In ESMP, relaxation of H⁺ exhibited two first-order phases, both an expression of the H⁺ conductivity of the ATPase complex. The rapid phase results from transient enhancement of H⁺ conduction caused by respiratory H⁺ itself. Oligomycin,N,N-dicyclohexylcarbodiimide, and tetranitromethane inhibited both phases of H⁺ release, and butanedione accelerated both. Phenylglyoxal inhibited principally the slow phase of H⁺ conduction. In USMP, H⁺ release followed simple first-order kinetics. Oligomycin depressed H⁺ release, enhanced respiratory H⁺, and restored the biphasicity of H⁺ release. Phenylglyoxal and tetranitromethane inhibited H⁺ release in USMP without modifying its first-order kinetics. Butanedione treatment caused biphasicity of H⁺ release from USMP, introducing a very rapid phase of H⁺ release. Addition of soluble F₁ to USMP also restored biphasicity of H⁺ release. A mechanism of proton conduction by F _o is discussed based on involvement of tyrosine or other hydroxyl residues, in series with the DCCD-reactive acid residue. There are apparently two functionally different species of arginine or other basic residues: those modified by phenylglyoxal, which facilitate H⁺ conduction, and those modified by butanedione, which retard H⁺ diffusion.     

Rabit distal colon epithelium: II. Characterization of (Na+, K+, Cl−)-cotransport and [3H]-bumetanide binding

Summary Loop diuretic-sensitive (Na⁺,K⁺,Cl^–)-cotransport activity was found to be present in basolateral membrane vesicles of surface and crypt cells of rabbit distal colon epithelium. The presence of grandients of all three ions was essential for optimal transport activity (Na⁺,K⁺) gradien-driven³⁶Cl fluxes weree half-maximally inhibited by 0.14 m bumetanide and 44 m furosimide. While⁸⁶Rb uptake rates showed hyperbolic dependencies on Na⁺ and K⁺ concentrations with Hill coefficients of 0.8 and 0.9, respectively, uptakes were sigmoidally related to the Cl^– concentration, Hill coefficient 1.8, indicating a 1 Na⁺: 1 K⁺:2 Cl^– stoichiometry of ion transport.The interaction of putative (Na⁺, K⁺, Cl^–)-cotransport proteins with loop diuretics was studied from equilibrium-binding experiments using [³H]-bumetanide. The requirement for the simulataneous presence of Na⁺,K⁺, and Cl^–, saturability, reversibility, and specificity for diuretics suggest specific binding to the (Na⁺, K⁺, Cl^–)-cotransporter. [³H]-bumetanide recognizes a minimum of two classes of diuretic receptors sites. high-affinity (K _D1=0.13 m;B _max1 =6.4 pmol/mg of protein) and low-affinity (K _D2=34 m;B _max2=153 pmol/mg of protein) sites. The specific binding to the high-affinity receptor was found to be linearly competitive with Cl^– (K ₁=60mm), whereas low-affinity sites seem to be unaffected by Cl^–. We have shown that only high-affinity [³H]-bumetanide binding correlates with transport inhibition raising questions on the physiological significance of diuretic receptor site heterogeneity observed in rabbit distal colon epithelium.     

Reactivation of photodynamically inactivated lambda phages

Summary UV-irradiated c phages show a lower survival when plated on rec ^–-cells as compared to rec ⁺-cells. Photodynamically inactivated c phages show the lowest survival on hcr ^–; rec ^–-cells. The rec-functions do not influence the repair of UV- or photodynamically induced lesions in T1 phages.     

Osmosensitivity of the 2H+−K+-exchange and the H+-ATPase complex F0F1 in anaerobically grownEscherichia coli

Escherichia coli grown anaerobically for osmotic studies upon increased osmolarity in alkaline medium carried out H⁺–K⁺-exchange in two steps, the first of which was DCCD¹ sensitive and osmo-dependent and had the 2H⁺/K⁺ stoichiometry. H⁺-efflux in the presence of protonophore (CCCP) upon increase of osmolarity was shown to be high and inhibited by DCCD, whereas H⁺-efflux induced by a decrease of osmolarity was small and not inhibited by DCCD. The 2H⁺/K⁺-exchange was absent intrkA anduncA mutants. InuncB mutant 2H⁺/K⁺-exchange was not DCCD-and osmosensitive. Competition between DCCD and osmoshock on inhibition of 2H⁺/K⁺-exchange was found. Osmosensitivity of this exchange disappeared in spheroplasts. Osmosensitivity of both 2H⁺/K⁺-exchange and the F₀F₁ and osmoregulation of the F₀F₁ via F₀ and a periplasmic space are postulated.Abbreviations F₀F₁ H⁺-ATPase complex - F₀ H⁺-channel, proteolipid - F₁ H⁺-ATPase - Trk constitutive system for K⁺ uptake - PV periplasmic protein valve - DCCD N,N-dicyclohexylcarbodiimide - CCCP carbonylcyanide-m-chlorophenylhydrazone - _H or _K transmembrane electrochemical gradient for H⁺ or K⁺ respectively - membrane potential - upshock or downshock increase or decrease of medium osmolarity, respectively - CGSC E. coli Genetic Stock Center, Yale University, USA