Chloroplast transfer in Nicotiana based on metabolic complementation between irradiated and iodoacetate treated protoplasts

Protoplasts of a light sensitive plastome mutant of Nicotiana tabacum (2 n=48) were irradiated and fused with iodoacetate-treated Nicotiana plumbaginifolia (2 n=20) protoplasts. Treated parental protoplasts were unable to divide. Metabolic complementation, however, helped the recovery of interspecific fusion products which survived and formed calli. Altogether 40 clones were investigated. N. plumbaginifolia plants were obtained in 15 clones (38%), somatic hybrids in 23 clones, and both types of regenerates were found in 2 clones. Irradiation therefore significantly increased the frequency of segregant formation with the non-irradiated N. plumbaginifolia nuclei (the frequency was 1.4% in the absence of irradiation). Regenerated plants in most cases (31 out of 34) contained chloroplasts from the irradiated parent. In 6 clones plants were obtained with both types of chloroplast. Thus, irradiated N. tabacum chloroplasts had an improved chance of dominating the heterokaryonderived cells, many of which contained N. plumbaginifolia nucleus. The system described should be generally applicable for the transfer of chloroplasts without the use of selectable genetic markers.     

Identification of Escherichia coli genes whose expression increases as a function of external pH

Summary Using transposon TnphoA and a plate screening method, we have isolated a set of Escherichia coli strains carrying phoA fusions with genes whose expression is modulated as a function of external pH. Besides fusions with the ompF gene and the malB locus, thirteen independent fusions were analysed whose expression is maximal during growth at pHs ranging from 7.0 to 8.5 and minimal during growth at pH 5.0. Six different genetic loci, called phmA, phmB, phmC, phmD, phmE and phmF (for pH modulated) were characterized and localized on the E. coli chromosome at approx. 12, 18, 41, 45, 75 and 84 min, respectively. Expression of phmA: :phoA fusions is also influenced when internal pH or environmental conditions such as osmolarity or anaerobiosis are modified. EnvZ protein is not involved in the regulation of phm : :phoA fusions.     

Tomato cybrids with mitochondrial DNA from Lycopersicon pennelli

Summary Cybrids have been regenerated following protoplast fusion of iodoacetamide-treated leaf mesophyll cells of Lycopersion esculentum cv UC82 and gamma-irradiated cell suspensions of L. pennellii, LA716. The cybrids were recovered in the regenerant population at a frequency of 19%, no selection pressure was applied for the persistence of the donor cytoplasm. The nuclear genotype of ten cybrids was characterized extensively using isozyme markers, cDNA-based restriction fragment length polymorphisms (RFLPs), and the morphology of the plants. No nuclear genetic information from L. pennellii was detected in the cybrids. The organellar genotype of the cybrids was determined using cloned probes and species-specific RFLPs. All the cybrids had inherited the tomato chloroplast genome and had varying amounts of L. pennellii mitochondrial DNA. The cybrids all had a diploid chromosome number of 24, produced pollen, and set seed.     

Analysis of cytoplasmic genomes in somatic hybrids between navel orange (Citrus sinensis Osb.) and ‘Murcott’ tangor

Summary Somatic hybrid plants were produced by protoplast fusion of navel orange and Murcott tangor. Hybridity of the plants was confirmed by the restriction endonuclease analysis of nuclear ribosomal DNA. All of the plants (16 clones) were normal, uniform, and had the amphidiploid chromosome number of 36 (2n=2x=18 for each parent). The cpDNA analysis showed that each of the 16 somatic hybrids contained either one parental chloroplast genome or the other. In all cases, the mitochondrial genomes of the regenerated somatic hybrids were of the navel orange type.Contribution No. E-132 of the Fruit Tree Research Station     

Assembly and sealing of tight junctions: Possible participation of G-proteins,phospholipase C,protein kinase C and calmodulin

Summary The making and sealing of a tight junction (TJ) requires cell-cell contacts and Ca^2–, and can be gauged through the development of transepithelial electrical resistance (TER) and the accumulation of ZO-1 peptide at the cell borders. We observe that pertussis toxin increases TER, while AIF₃ and carbamil choline (carbachol) inhibit it, and 5-guanylylimidodiphosphate (GTPs) blocks the development of a cell border pattern of ZO-1, suggesting that G-proteins are involved. Phospholipase C (PLC) and protein kinase C (PKC) probably participate in these processes since (i) activation of PLC by thyrotropin-1 releasing hormone increases TER, and its inhibition by neomycin blocks the development of this resistance; (ii) 1,2-dioctanoylglycerol, an activator of PKC, stimulates TER development, while polymyxin B and 1-(5-isoquinoline sulfonyl)-2-methyl-piperazine dihydrochloride (H7), which inhibit this enzyme, abolish TER. Addition of 3-isobutyl-1-methyl-xanthine, dB-cAMP or forskolin do not enhance the value of TER, but have just the opposite effect. Trifluoperazine and calmidazoline inhibit TER development, suggesting that calmodulin (CaM) also plays a role in junction formation. These results indicate that junction formation may be controlled by a network of reactions where G-proteins, phospholipase C, adenylate cyclase, protein kinase C and CaM are involved.     

Lipid-protein surface films generated from membrane vesicles: selfassembly,composition, and film structure

Lipid-protein films at the air-water interface were generated from a variety of native vesicles and from vesicles derived from lipid extracts. A technique is described which is particularly suitable for the generation of films from small amounts of material at high yield and velocity. In all instances, 10 l vesicle suspensions containing 25 g protein yield at least 50 cm² film area at a constant surface pressure of 12 mN/m within minutes. Upon formation, surface films are separated from vesicles by use of shear forces. Complete separation is demonstrated by electron microscopy and surface pressure-area diagrams. The latter confirms previous conclusions that surface films generated from lipid vesicles are organized as a monolayer. Analysis of lipid-protein surface layers reveals that their lipid to protein ratios match those of the vesicles used, within a factor of two, irrespective of whether films are generated at high or low surface pressure. Surface denaturation of membrane proteins is shown to be effectively prevented when the film is generated and held at high surface pressure ( 15 mN/m). Upon surface pressure jumps from high to low values, denaturation kinetics revealed activation areas of 1.5 (±0.2) nm². Offprint requests to: H. Schindler     

Thrombin and H64A subtilisin cleavage of fusion proteins for preparation of human recombinant parathyroid hormone

Human parathyroid hormone, hPTH, an 84 amino acid polypeptide, was produced intracellularly inEscherichia coli as a fusion protein, linked to the C-terminus of a 15 kD IgG-binding protein. Approximately 100 mg fusion protein was obtained per liter fermentation medium. To test the efficiency of two alternative enzymatic cleavage methods, two fusion proteins differing only in the linker region were constructed. Cleavage of a Phe-Phe-Pro-Arg linker was obtained with bovine thrombin and cleavage of a Phe-Ala-His-Tyr linker with recombinant H64A subtilisin. Both enzmes yielded the correct N-terminus and cleaved their respective linkers quantitatively, although additional internal cleavage sites in hPTH were detected and characterized. The linker cleavage conditions were optimized and hPTH was purified to homogeneity. Thrombin cleavage resulted in a final yield of 5 mg hPTH/L, while H64A subtilisin cleavage was more specific and gave 8 mg/L. The purified recombinant product was identical to native hPTH and exhibited full biological activity in an adenylate cyclase assay.     

Induction of cell fusion by a factor released by the cellular slime mold Polysphondylium pallidum

Heterokaryons and hybrid cells, which are extremely useful for research in cell biology, can be produced artificially by treating cells with either polyethylene glycol or certain inactivated viruses that alter the plasma membrane. We report here a novel cell-fusion inducing factor secreted by CK-8 strain cells of cellular slime mold Polysphondylium pallidum. Treatment of other strains or other species of cellular slime molds, such as NC-4 of Dictyostelium discoideum with the diluted fraction, containing molecules larger than 50 kDa, of the conditioned medium of CK-8 cell culture induces cell fusion at high frequency and produces multinucleated large cells. This cell fusion is inducible between cells of either a single strain or of two different strains of cellular slime molds.Abbreviations BSS Bonner's salt solution - CM conditioned medium - EDTA ethylenediaminetetraacetic acid - F2 fraction containing cell-fusion induction factor - Mr molecular mass     

Intersectional cytoplasmic hybrids in Nicotiana

Summary Cybrid plants having the nuclear genomes of one species and either or both plastomes and chondriomes of another species were obtained by fusing protoplasts of Nicotiana sylvestris, as recipients, with X-irradiated protoplasts of N. rustica as donors of chloroplasts and mitochondria. Forty-nine flowering plants, derived from 28 calli, were analysed. As expected, they all had N. sylvestris (i.e. recipients) morphology. Chloroplast DNA restriction patterns indicated that 8 and 41 plants had N. rustica and N. sylvestris plastomes, respectively. Some of the plants with either type of plastomes produced sterile pollen but none showed anther malformation typical to alloplasmic male sterility. Chondriome identification by mitochondrial DNA restriction analysis of cybrid plants revealed only restriction patterns which were either similar or identical to those of N. sylvestris while no cybrids with N. rustica restriction patterns were detected.     

Regulation of E. coli phenylalanyl-tRNA synthetase operon in vivo

The phenylalanyl-tRNA synthetase operon is composed of two adjacent, cotranscribed genes, pheS and pheT, corresponding respectively to the small and large subunit of phenylalanyl-tRNA synthetase. A fusion between the regulatory regions of phenylalanyl-tRNA synthetase operon and the lac structural genes has been constructed to study the regulation of the operon. The pheS,T operon was shown, using the fusion, to be derepressed when phenylalanine concentrations were limiting in a leaky auxotroph mutated in the phenylalanine biosynthetic pathway. Furthermore, a mutational alteration in the phenylalanyl-tRNA synthetase gene, bradytrophic for phenylalanine, was also found to be derepressed under phenylalanine starvation. These results indicate that the pheS,T operon is derepressed when the level of tRNA^Phe aminoacylation is lowered. By analogy with other well-studied amino acid biosynthetic operons known to be controlled by attenuation, these in vivo results indicate that phenylalanyl-tRNA synthetase levels are controlled by an attenuation-like mechanism.