Enhanced competitiveness for nodulation of Medicago sativa by Rhizobium meliloti transconjugants harbouring the root-inducing plasmids of Agrobacterium rhizogenes strain 15834

Abstract The root-inducing plasmid of Agrobacterium rhizogenes strain 15834 marked with transposon Tn5-mob with a helper plasmid RP4-4 was mobilized into nitrogen-fixing Rhizobium meliloti strain CIAM 1759. The resulting transconjugants did not induce ‘hairy’ root syndrome but developed nitrogen-fixing nodules on alfalfa. Among the 9 transconjugants tested, 6 strains had increased nodulation rates. The competitiveness of 2 of these R. meliloti (pRi) strains was significantly enhanced as compared with the parent strain CIAM 1759; this was confirmed both in tube and in pot tests.     

Exocytotic fusion pores exhibit semi-stable states

Summary Rapid-freezing/freeze-fracture electron microscopy and whole-cell capacitance techniques were used to study degranulation in peritoneal mast cells of the rat and the mutant beige mouse. These studies allowed us to create a time-resolved picture for fusion pore formation. After stimulation, a dimple in the plasma membrane formed a small contact area with the secretory granule membrane. Within this zone of apposition no ordered proteinaceous specializations were seen. Electrophysiological technique measured a small fusion pore which widened rapidly to 1 nS. Thereafter, the fusion pore remained at semi-stable conductances between 1 and 20 nS for a wide range of times, between 10 and 15,000 msec. These conductances correspond to pore diameters 25–36 nm. Ultrastructural data confirmed small pores of hourglass morphology, composed of biological membrane coplanar with both the plasma and granular membranes. Later, the fusion pore rapidly increased in conductance, consistent with the observed morphology of omega-figures. The hallmarks of channel-like behavior, instantaneous jumps in pore conductance between defined levels, and sharp peaks in histograms of conductance dwell-time, were not seen. Since the morphology of small pores shows contiguous fracture planes, the electrical data represent pores that contain lipid. These combined morphological and electrophysiological data are consistent with a lipid/protein complex mediating both the initial and later stages of membrane fusion.We would like to dedicate this paper to the memory of our friend and mentor, Alex Mauro, who emphasized to us the importance of equivalent circuits. This work was supported by National Institutes of Health grant GM-27367, and National Science Foundation grant IBN-91117509.     

The effects of an Escherichia coli dnaAts mutation on the replication of the plasmids colE1 pSC101, R100.1 and RTF-TC.

Summary The rate of replication of the plasmids colE1, pSC101, R100.1 and pAR132 (an RTF-TC derivative of the drug resistance factor R100.1) has been investigated directly by DNA: DNA hybridization. These rates have been compared, in a dnaAts strain, to that of various markers of the host chromosome at permissive and non-permissive temperatures. Chromosome initiation in the dnaAts strain stops rapidly after a shift to the non-permissive temperature, but plasmids R100.1 and pAR132 do not seem to be affected directly and continue replication for some time. The colE1 replication rate undergoes a large increase after the temperature shift, followed by a rapid decrease to a very low level 25 min after the shift. In contrast pSC101 replication stops immediately after the shift. ColE1 is able to replicate in an integratively suppressed dnaAts strain at 42° C whereas pSC101 stops replication immediately under these conditions. We conclude that R100.1 and its derivative RTF-TC can replicate without a functional dnaA product; that colE1, while affected by a shift in temperature in a dnaAts strain, does not directly require dnaA; and that the plasmid pSC101 has an absolute requirement for dnaA. The absolute requirement of pSC101 for dnaA in the integratively suppressed Hfr strain provides a useful system for further investigations of the dnaA function.     

Accelerated breakdown of reticulocyte protein formed under conditions of amino acid depletion.

1. Labile protein is formed when rat or rabbit reticulocytes are incubated in medium deficient in individual amino acids, especially histidine, valine or alanine. The fraction of unstable protein is increased to about 35% of the total protein synthesized when the histidinyl-tRNA-charging inhibitor, histidinol, is added to histidine-deficient media. 2. The molecular weights of the labile proteins measured by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence of urea are less than haemoglobin and probably represent prematurely terminated haemoglobin chains. 3. Although protein synthesis is always lower under conditions that produce labile protein, inhibition of protein synthesis by fluoride or cycloheximide does not give an effect similar to amino acid depletion. 4. The synthesis of protein in deficient medium does not alter the degradation rate of pre-existing protein in reticulocytes and is thus unrelated to the stringent response in bacteria. 5. We propose that amino acid-deficient medium leads to a decreased charging of the appropriate tRNA, a concomitant decrease in protein synthesis and the degradation of nascent peptides.     

Intracellular uptake and α-amylase and lactate dehydrogenase releasing actions of the divalent cation ionophore A23187 in dissociated pancreatic acinar cells

Summary Intracellular uptake of A23187 and the increased release of amylase and lactate dehydrogenase (LDH) accompanying ionophore uptake was studied using dissociated acinar cells prepared from mouse pancreas. Easily detected changes in the fluorescence excitation spectrum of A23187 upon transfer of the ionophore from a Tris-buffered Ringer's to cell membranes were used to monitor A23187 uptake. Uptake was rapid in the absence of extracellular Ca²⁺ and Mg²⁺ (t1/2=1 min) and much slower in the presence of Ca²⁺ or Mg²⁺ (t1/2=20 min). Cell-associated ionophore was largely intracellular as indicated by fluorescence microscopy, lack of spectral sensitivity to changes in extracellular Ca²⁺ and Mg²⁺, and by equivalent interaction of ionophore with membranes of whole and sonicated cells.A23187 (10 m) increased amylase release 200% in the presence of extracellular Ca²⁺ and Mg²⁺. In the absence of Ca²⁺ (but in the presence of Mg²⁺) A23187 did not increase amylase release. A23187 (10 m) also produced Ca²⁺-dependent cell damage, as judged by increased LDH release, increased permeability to trypan blue, and by disruption of cell morphology. The cell damaging and amylase releasing properties of A23187 were distinguished by their time course and dose-response relationship. A23187 (1 m) increased amylase release 140% without increasing LDH release or permeability to trypan blue.     

Cytotoxic T-cell responses to H-Y:Ir genes and associative antigens map inH-2

The secondary cytotoxic responses to the male-specific antigen (H-Y) in mice showH-2 restriction so that the cytotoxic female cell must share the K- and/or D-end antigen with the male target cells. The association with the K and/or D end varies with differentH-2 haplotypes,e.g., H-2 ^b cytotoxic cells require the H-2D^b antigen(s) on the target cells, while cytotoxic cells fromH-2 ^b/H-2 ^d F₁ mice sensitized toH-2 ^d male cells kill only male targets having H-2K^d antigen(s). This association of H-Y with appropriate K/D antigens seems to be needed also in the induction of the cytotoxic response. Of the independent haplotypes, onlyH-2 ^b strains are capable of making secondary anti-H-Y responses and this trait seems to be dominant,i.e., the F₁ strains with oneH-2 ^b parent are able to produce anti-H-Y cytotoxic cells against both theH-2 ^b parent and the nonresponder parent. The mating of the two nonresponder strains may produce F₁ mice which are responders, thus suggestingIr gene complementation. Mapping data indicates that at least one of these complementary genes is located in theI-C region fork/s complementation.     

Suppression of an Escherichia coli dnaA mutation by the integrated R factor R100.1: origin of chromosome replication during exponential growth.

We have investigated the behavior, during exponential growth, of strains of Escherichia coli carrying a dnaA(Ts) mutation that has been suppressed by the integration of the F-like R plasmid R100.1. We present evidence showing that replication in these strains proceeds largely from the normal chromosome origin at 30 degrees C, a permissive temperature for the dnaA(Ts) gene product, whereas, at 42 degrees C, replication proceeds largely from the integrated plasmid. These conclusions are based on measurements made by deoxyribonucleic acid:deoxyribonucleic acid hybridization of the relative frequencies of the prophages Mu-1 and lambdaind- and R100.1 integrated at known locations on the E. coli chromosome in these Hfr strains.     

Experimental histoplasmosis capsulati in athymic nude mice

Granuloma formation in nude (nu/nu) mice and their heterozygous littermates (nu/+ mice) against Histoplasma capsulatum var. capsulatum infection was studied.A culture of H. capsulatum var. capsulatum, isolated from a granuloma in the nasal cavity of a Japanese patient, was used in this experiment. Sixteen specific-pathogen-free male nu/nu and 32 nu/+ mice were used in this study.The nu/+ mice were divided into two groups. Sixteen nu/+ mice in one group and 16 nu/nu mice were inoculated intraperitoneally with 10⁶ yeast cells of the fungus, those in the other group of nu/+ mice were inoculated intravenously with the same number of the yeast cells. Two mice out of each group were sacrificed 2, 3, 7, 11, 14, 18, 25 and 30 days after inoculation, and each of their organs was examined histopathologically. In addition, pieces of these tissues were cultured on Sabouraud's dextrose agar slants.In the nu/+ mice inoculated intraperitoneally, although the fungus was recovered from the spleen, kidney and lymph nodes during the initial course of the infection, lesions were not detected in their histopathological sections. In the nu/+ mice inoculated intravenously, colonies were recovered from all of the organs examined, other than the brain and thymus, 7 days after inoculation.Histopathologically, a few microfoci consisting chiefly of mononuclear cells with or without yeast cells were found in the liver sections 4 days after inoculation. Seven and 11 days after inoculation the number of lesions had increased. They had large accumulations of mononuclear cells. From day 14 on, almost all of the yeast cells had lost most of their staining affinity or were destroyed in the granuloma. From day 25 on, the granulomatous lesions changed gradually to fibrous tissue.In the nu/nu mice the fungus was readily recovered from the spleen, liver, kidney and lymph nodes. Histopathologically, a few microfoci consisting of mononuclear cells were present in the liver sections 4 days after inoculation. That is to say, during the initial course of infection granulomas were formed. In the liver, from day 7 on, the lesions were large and their number increased. However, there was a definite difference between the nu/nu and nu/+ mice. In the former, the yeast cells were not killed, and they continued to multiply within the granulomas. These granulomas were never transformed into fibrous tissue.     

γ-Glutamyl Cyclotransferase: A New Genetic Polymorphism in the Mouse (MUS MUSCULUS) Linked to LYT–2

Electrophoretically variant forms of gamma-glutamyl cyclotransferase have been identified in red cells of inbred mouse strains. Each inbred strain exhibited a major band of activity and a minor band that migrated more anodally. The polymorphism affects the migration of both the major and minor bands in a similar way. F1 hybrids between strains with fast forms (A/J) and strains with the slow forms (C57BL/6J) exhibited a four-banded pattern consistent with co-dominant inheritance. The patterns observed in backcross and F2 mice were consistent with the segregation of a pair of autosomal co-dominant alleles. Recombinant inbred strains and a congenic strain were used to show that the locus controlling gamma-glutamyl cyclotransferase (Ggc) is linked to Lyt-2, a lymphocyte alloantigen locus on chromosome 6, with an estimated map distance of 5.0 +/- 2.5 centimorgans.