Transmission of paternal chloroplasts in Nicotiana

Summary Transmission of paternal chloroplasts was observed in Nicotiana, considered to inherit organelles in a strictly maternal way. Plants carrying streptomycin resistant plastids were used as pollen donors. Cell lines with paternal plastids in the offspring were selected as green (resistant) sectors on calli induced from the seedlings on streptomycin-containing media. The presence of paternal plastids in the regenerated plants was confirmed by restriction analysis. In the Nicotiana plumbaginifolia xN. plumbaginifolia Np(SR1)3 and the N. plumbaginifolia Np(gos)29 xN. tabacum SR1 crosses 2.5% and 0.07% of the offspring were found to contain paternal (tabacum) plastids, respectively. These plants, however, carried maternal mitochondria exclusively. This sexual cybridization method offers a simple way to transfer chloroplasts solely, a goal not accessible by protoplast fusion.     

Direct selection of cybrids by streptomycin and valine resistance in tobacco

Summary Direct selection of cybrids by simultaneous selection for donor chloroplasts and for the recipient nuclei is described. Mesophyll protoplasts of two tobacco (Nicotiana tabacum) mutants, SR1 (streptomycin resistant) and Val^r-2 (valine resistant), were fused by polyethylene glycol treatment. Streptomycin resistance in the SR1 mutant is a maternally inherited chloroplast trait while valine resistance is a Mendelian (nuclear) digenic recessive character. The fused protoplast population was cultured and colonies were selected for resistance to valine (1 mM) and streptomycin (343 M). The efficiency of selection has been confirmed in three clones by demonstrating seed transmission of both streptomycin and valine resistances. In one subclone both streptomycin resistant and sensitive plants were obtained indicating that the streptomycin sensitive chloroplasts had not been totally eliminated by growth on the selective medium.     

Isolation and characterization of streptomycin-resistant mutants in Nicotiana plumbaginifolia

Summary Streptomycin-resistant colonies were isolated from protoplast cultures of haploid Nicotiana plumbaginifolia based on their ability to green in medium containing 1 mg/ml streptomycin sulfate. The frequency of resistant colonies was 0.9×10^–5 in nonmutagenized culture, and increased ten-fold following treatment of culture with 10 g/ml N-methyl-N-nitro-N-nitrosoguanidine. Of a total of 52 resistant clones isolated, 2 gave rise to haploid, 15 to diploid, and 3 to tetraploid plants upon transfer of calli to differentiation medium. Leaf-segment and protoplast assays showed that all diploid regenerates were resistant to streptomycin but sensitive to chloramphenicol, kanamycin, lincomycin, neomycin, and spectinomycin. Plants in most diploid clones were fertile and able to set seeds when self-fertilized and crossed reciprocally to wild-type plants. Inheritance of streptomycin resistance was studied in the diploid clones and, without exception, the resistance was transmitted maternally. Comparative studies of the ultrastructure of organelles and protein synthesis in isolated chloroplasts between wild-type and resistant clones in the presence of streptomycin suggest that streptomycin resistance is controlled by chloroplasts.     

Plant regeneration from cytoplasmic hybrids of rice (Oryza sativa L.)

Summary We obtained cybrid plants by electrofusing -irradiated protoplasts of a cytoplasmic male-sterile line A-58 CMS (Oryza sativa L.) and iodoacetamide (IOA)-treated protoplasts of the fertile (normal) rice cultivar Fujiminori. The cybridity of the plants was confirmed by mitochondrial (mt) DNA restriction endonuclease, and plasmid-like DNA analyses, and by isozyme, cytological and morphological investigations. The chromosome number of the cybrid plants is 24.     

Freeze-fracture studies on the cockroach hemocyte membrane complex: Symmetric and asymmetric membrane particle distributions

Summary Freeze-fracture studies were conducted on the membranes of normal cockroach hemocytes. The plasmalemma is asymmetric with the A fracture face containing 80–100 Å membrane intercalated particles at a concentration of 2500/². The B fracture face contains 120–150 Å particles with a relatively low density (800/²). The nuclear envelope displays an asymmetry with the A fracture face containing 1500 particles/² and the B face containing 300/ ². No significant particle size differences were observed in nuclear envelope fracture faces. Two types of symmetric membranes were also found in these cells. Both A and B fracture faces of the membrane surrounding the numerous cytoplasmic inclusion bodies contain particle sizes and concentrations similar to the B face of the plasmalemma. A second type of symmetry was observed in cells apparently engaged in exocytosis. Vesicles (0.1 D) from this process were completely particle free on both fracture faces. Such particle free vesicles could be found in the cytoplasm, attached to the plasmalemma, or completely separated from the cell.Supported by a Pharmaceutical Manufacturers Association Foundation Fellowship.The author wishes to thank Ms. Annalena K. Charla for assistance in plate preparation, Dr. Julius Schultz and the Papanicolaou Cancer Research Institute for use of the freeze-etch device, and Dr. David Smith for the electron microscope facilities.     

The chromosome idiogram of Nicotiana plumbaginifolia

The karyotype of Nicotiana plumbaginifolia (2n=20) was determined by the analysis of 25 metaphase plates of both haploid and diploid plantlets. The material originated from root tip meristems and leaf protoplasts. Chromosome length ranged from 1.73 to 4.34 m and the total length of the genome was 30.2 m. The following chromosome pairs have been recognized: three metacentrics, two submetacentrics, one subtelocentric, one subtelocentric with satellites and three telocentrics.Contribution no 2036 of the Biology, Radiation protection and Medical Research Programme, Directorate General XII of the Commission of the European Communities.     

Some observations on the fine structure of the myotendinous junction in myotomal muscles of the tadpole tail

Summary Myotendinous junctions in the myotomal tail muscles of the tadpole of Rana rugosa were examined by electron microscopy. At the site of the myotendinous junction, the sarcolemma is covered on its sarcoplasmic aspect by the connecting filament layer and the attachment layer, and on the extracellular aspect by the intermediary layer and the external lamina, with associated collagen fibrils. The intermediary layer consists of filamentous structures which closely resemble microfibrils (Hanak and Böck, 1971), spine-like or thread-like profiles (Korneliussen, 1973) and intermediary layer (Nakao, 1975a, b) in the myotendinous junctions of other vertebrate skeletal muscles.Particularly interesting is the fact that all the coverings and linings of the sarcolemma, including the external lamina, are completely absent in the terminal segment of the finger-like sarcolemmal invagination characteristic of the myotendinous junction. Furthermore, special types of coupling between a sac of sarcoplasmic reticulum and a part of the sarcolemmal invagination are frequently observed. These couplings always occur along the region of the sarcolemma where the external lamina is absent. The couplings show features similar to those of the triad, such as SR feet , scalloped SR membranes and granular content of the SR sac, suggesting that they are analogous and functionally similar to the triad and other equivalent structures.     

Requirements for in vitro propagation of seven nitrogen-fixing Alnus species

Studies on the in vitro propagation of Alnus crispa, A. glutinosa, A. incana, A. japonica, A. rubra, A. sinuata and A. viridis indicated interspecific as well as intraspecific variations in their requirements for in vitro culture. The WPM and Blaydes media supported, respectively, growth of A. glutinosa and A. crispa but not that of both species, while the MS medium induced equal or significantly better growth than WPM and Blaydes media for both species. The optimum type and concentration of sugar to be used in the multiplication medium varied with species. Only A. glutinosa showed good growth on sucrose while glucose was optimum for all other species but at different concentrations. All species rooted in 3 weeks on half-strength MS medium including 1 M IBA. All clones of A. glutinosa and A. rubra rooted 100%, whereas easy-to-root and difficult-to-root clones were observed in the other species. In the rooting medium, glucose promoted rooting of the difficult-to-root clones better than sucrose. Survival following transfer to an artificial substrate was 100% for all species. Nodulation tests using pure cultures of two Frankia strains showed 100% nodulation on all Alnus clones.     

Enhancement of β-glucosidase stability and cellobiose-usage using surface-engineered recombinant Saccharomyces cerevisiae in ethanol production

To enhance the use of cellobiose by a recombinant Sachharomyces cerevisiae, the expressed -glucosidase that hydrolyzes cellobiose was stabilized using a surface-display system. The C-terminal half of -agglutinin was used as surface-display motif for the expression of -glucosidase in the cell wall. The surface-displayed -glucosidase had a half-life time (t _1/2) of 100 h in acidic culture broth conditions, while secreted -glucosidase had a t _1/2 of 60 h. With such stabilization of -glucosidase, the surface-engineered S. cerevisiae utilized 7.5 g cellobiose l^–1 over 60 h, while S. cerevisiae secreting -glucosidase into culture broth used 5.8 g cellobiose l^–1 over the same period.     

Streptomycin resistance is inherited as a recessive Mendelian trait in a Nicotiana sylvestris line

Summary The SR180 cell line has been isolated in a callus culture derived from a haploid Nicotiana sylvestris (n = X = 12) plant by its ability to proliferate on a selective medium containing 2,000 g/ml streptomycin sulphate. From the cell line diploid plants have been regenerated. The SR180 selfs are resistant to streptomycin. Streptomycin sensitivity in F1, and a 31 (sensitive to resistant) segregation in F2 indicate that resistance in the SR180 mutant is the result of a recessive Mendelian mutation.     

Photoreversion und Photoprotektion bei UV-induzierten Mutationen des nichtphotoreaktivierbaren Bacteriums E.coli phr?

Summary Stationary phase cells of strain phr^–/MC 2 ofE. coli are not photoreactivable but the frequency of UV-induced mutations to low Streptomycine-resistance (S 3, 3/ml) is decreased strongly by illumination with light of fluorescence tubes (310 to 500 nm) after UV-irradiation. Also dark-reversion (DRM) of these mutations due to keeping UV-irradiated cells in saline is observed. Illumination before UV-irradiation decreases the frequency of the mutations (photoprotection against mutation=PPM) to the same extent as the combined action of photoreversion (PRM) and DRM. The lag-phase of cell division is prolonged strongly by illumination from 80 min without light to 150 min by the light-dose of highest activity. The additional lag is nearly the same if the illumination is done before, after or without UV-irradiation; this lag is about additive to the small lag caused by UV. Pre-illumination of the stationary-phase cells does not cause photoprotection against killing (PP), it even decreases the survival after high UV-doses. The observations support the hypothesis that PRM in this strain may be indirect, i.e. caused by the light-induced additional division lag which enhances the dark repair of UV-premutations. Also spontaneous premutations which are apparently present in the stationary-phase cells seem to be influenced by the light in this way.     

6-Methylpurine-induced inhibition of sclerotia morphogenesis in Sclerotium rolfsii and its reversal by adenosine

In liquid synthetic medium inoculated with Sclerotium rolfsii (SR), addition of 6-methylpurine (MP, 50g/ml) immediately after inoculation led to approximately 100% reduction in sclerotia production. Adenosine, and to a lesser extent guanosine, each at final concentration of 100g/ml significantly reduced inhibition of sclerotia formation by SR in presence of 50g/ml MP. Uridine and cytidine each at 100g/ml had no such effect. The inhibition of sclerotia morphogenesis could be prevented by addition of 800g/ml of adenosine together with 50g/ml MP. Reversal by adenosine of MP-induced inhibition of sclerotia development was concentration dependent.     

Evidence of β-carotene 7,8(7′,8′) oxygenase (β-cyclocitral,crocetindial generating) in Microcystis

A -carotene oxygenase is described which occurs in the Cyanobacterium Microcystis. It cleaves -carotene and zeaxanthin specifically at the positions 7,8 and 7,8, while echinenone and myxoxanthophyll are not affected. The oxidative cleavage of -carotene leads to the formation of -cyclocitral and crocetindial and that of zeaxanthin to hydroxy--cyclocitral and crocetindial in nearly stoichiometric amounts. Oxidant is dioxygen as has been demonstrated by high incroporation (86%) of ¹⁸O₂ into -cyclocitral. -Carotene oxygenase is membrane bound, sensitive to sulfhydryl reagents, antioxidants and chelating agents. Iron seems to be an essential part of the enzyme activity. Cofactors necessary for the reaction could not be detected.Abbreviations TLC thin layer-chromatography - PIPES piperazine-N,N-bis-(2-ethanesulfonate) Na - TES 2{[tris-(hydroxymethyl)-methyl]-amino} ethanesulfonic acid Dedicated to Professor G. Drews on occasion of his 60th birthday     

Transformation of steroids by fungal protoplasts

Summary Protoplasts of Cunninghamella elegans transformed cortexolone to the same products as did the mycelium. Transformation of the steroid by non-induced mycelium and by protoplasts released from it was almost completely inhibited by cycloheximide. However, hydroxylation of cortexolone was not affected by this antibiotic if mycelium grown in the presence of an enzyme inducer or protoplasts obtained from the induced mycelium were used. The transformation rate of protoplasts, on the basis of dry weight or protein units, was about four times higher than that of the mycelium, indicating that the mycelial cell wall was a serious rate-limiting factor in steroid bioconversion.     

A third type of raphide crystal in the plant kingdom: Six-sided raphides with laminated sheaths in Agave americana L.

Summary Raphides in leaves of Agave americana L. have six-sided cross sections. Each crystal tapers off to a point at both ends. It is enveloped in a 100 nm thick sheath which, in cross section, shows lamellae with periods of 6–9 nm. No polysaccharides could be detected in the sheaths with the Thiéry reaction. Dissolution of a raphide in acid occurs slowly from both ends leaving the crystal sheath visible in the light microscope. The raphide cell walls contain a layer that in glutaraldehyde-fixed tissue reacts neither with the Thiéry stain nor with potassium permanagnate. Its morphology resembles the isotropic layer of Chafe and Chauret (Protoplasma 80, 129–147, 1974) but no lignification could be shown as yet. Though up to now only raphides with four-sided or H-shaped cross sections have been observed by electron microscopy, we suggest that many raphides described as rounded in light microscopy might in fact be six-sided.     

A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl β-d-lactoside derivatives

4-Methylumbelliferyl 6-O-benzyl--d-lactoside (6Bn-MU-Lac) and some related compounds were synthesizedvia different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, theK _m value is 0.232 mM at pH 5. Abbreviations: CGase, ceramide glycanase; Gal, galactose; Glc, Glucose; Lac, lactose; MU, 4-methylumbelliferone; MU-Lac, 4-methylumbelliferyl -d-lactoside; bBn-Lac, 6-O-benzyl-lactose; 6Bn-MU-Lac, 4-methylumbelliferyl 6-Obenzyl--d-lactoside; 46Bd-MU-Lac, 4-methylumbelliferyl 4,6-O-benzylidene--d-lactoside; MU-Cel, 4-methylumbellifery -d-cellobioside; 46Bd-MU-Cel, 4-methylumbelliferyl 4,6-O-benzylidene--d-cellobioside; TLC, thin layer chromatography;¹H-NMR, proton nuclear magnetic resonance; GSL, glycosphingolipids; CSA, 10-camphorsulfonic acid. See Scheme 1 for chemical structures.     

Effects of GABA antagonists,SR 95531 and bicuculline,on GABAA receptor-regulated chloride flux in rat cortical synaptoneurosomes

Synaptoneurosomes isolated from cerebral cortices of male Sprague-Dawley rats were used for studying GABA_A receptor-regulated chloride influx. The in vitro effects of GABA antagonists, SR 95531 (a pyridazinyl GABA derivative) and bicuculline, on pentobarbital-stimulated, muscimol-stimulated or flunitrazepam-enhanced, muscimol-stimulated chloride uptake were studied. The chloride uptake was determined at 30°C, for 5 sec. Pentobarbital and muscimol produced a maximal stimulation of chloride uptake in cortical synaptoneurosomes at 500 M and 50M, respectively. SR 95531 as well as bicuculline had no effect on the basal uptake of chloride. Whereas, SR 95531 (0.3–30 M) and bicuculline (0.1–100 M), when added 5 min before muscimol (50 M), produced a significant concentration-dependent inhibition of muscimol (50 M)-stimulated chloride uptake (IC₅₀ s of 0.89±0.11 M and 13.45±2.10M, respectively). In studies of the inhibitory effects of SR 95531 and bicuculline on pentobarbital (500 M)-stimulated chloride uptake, the IC₅₀ s were 0.81±0.12 M and 3.86±1.14 M, respectively. SR 95531 exhibited a more potent inhibitory effect than bicuculline on flunitrazepam-enhanced, muscimol-stimulated chloride uptake. The results revealed that SR 95531 has a more potent antagonistic effect than bicuculline on GABA_A-regulated chloride flux.     

Characterization of cDNA clones for rye endosperm β-amylase and analysis of β-amylase deficiency in rye mutant lines

Summary In order to characterize a -amylase deficiency in the endosperm of mutant rye lines, homologous cDNA probes were prepared. A rye cDNA library was constructed from a normal line and screened with a barley -amylase probe. Three partial cDNA clones specific for endosperm -amylase in rye were isolated and characterized. The largest of these clones was used to investigate the expression of endosperm -amylase in mutant and normal lines by Northern hybridization. These experiments, as well as in vitro translation experiments, demonstrate the absence of endosperm -amylase mRNA in mutant lines. Sequencing of three different cDNA clones revealed a single nucleotide difference, which suggests that two genes encoding endosperm -amylase genes might exist in rye. From Southern blots we anticipate that these two genes are tightly linked. Results of these experiments and previous data indicating that the mutation was located within the -amylase locus on chromosome 5 are consistent with the hypothesis that the mutation results from a deletion simultaneously affecting the two genes. However, due to extensive polymorphism within normal lines used as control, additional experiments will be required to further substantiate this conclusion. The deduced amino acid sequence reveals the occurrence of three short glycine-rich repeats containing 11 or 12 residues close to the carboxyl terminus of the protein. A comparison of the nucleotide sequence between rye and previously described barley cDNA clones revealed ca. 90% homology at the amino acid level, except in this C-terminal repeated part, where it drops to 45%.     

Resistance ofBradyrhizobium japonicum strains to selected antibiotics

The majority of the 50Bradyrhizobium japonicum strains tested were resistant to ampicillin, kanamycin, streptomycin and tetracycline in concentrations below 100 g/ml but resistant to chloramphenicol in concentrations equal to or above 100 g/ml. Two strains had high levels of resistance to ampicillin and to streptomycin and six strains were very sensitive to several antibiotics.

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Résumé La majorité de la cinquantaine de souches deBradyrhizobium japonicum s'est révélée résistante à l'ampicilline, à la kanamycine, à la streptomycine et à la tétracycline aux concentrations intérieures à 100g/ml mais au chloramphenicol aux concentrations égales ou supérieures à 100 g/ml. Deux souches présentalent un haut degré de résistance à l'ampicilline et à la streptomycine. Six souches étaient très sensibles à plusleurs antibiotiques.