Genetic transformation of flax (Linum usitatissimum) by Agrobacterium tumefaciens: regeneration of transformed shoots via a callus phase

Genetic transformation of flax (Linum usitatissimum) has been achieved using an A. tumefaciens strain carrying a non-oncogenic Ti plasmid-derived vector containing a chimaeric npt-II gene and a wild type nopaline synthase gene. Fertile, transformed shoots were most easily obtained from Km^r callus developing on hypocotyl sections. The totipotency of the Km^r callus was dependent upon its origin. T-DNA was visualised by Southern blotting in all Km^r tissues. Efficient expression of nopaline synthase and the chimaeric npt-II gene was found in transformed Km^r callus and regenerated shoots.Abbreviations npt-II neomycin phosphotransferase II gene - NPT-II neomycin phosphotransferase II - nos nopaline synthase gene promoter - Km^r kanamycin resistant - BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - MSD4×2 medium D4×2 based on Murashige & Skoog medium (see Scott & Draper, 1987)     

Distinct phases of the fluorescence response of the lipophilic probe N-phenyl-1-naphthylamine in intact cells and membrane vesicles of Escherichia coli

The fluorescence of the lipophilic prbe N-phenyl-1-naphthylamine (NPN) bound to intact cells of Escherichia coli is quenched by the addition of glucose, succinate, -lactate, pyruvate, formate and glycerol. Partial recovery of fluorescence occurs on anaerobiosis. Use of mutants with defects in the ATP synthase or the respiratory chain show that quenching of fluorescence may be energized either by ATP hydrolysis or by substrate oxidation through the respiratory chain. Permeabilization of the outer membrane by treatment of intact cells with EDTA, or use of a mutant with an outer membrane permeable to lipophilic substances, results in a more rapid binding of NPN and in a decrease in quenching observed on substrate addition. NPN binds rapidly to everted membrane vesicles, but does not respond to membrane energization. It is proposed that inner membrane energization in intact cells alters the binding or environment of NPN in the outer membrane. The fluorescence recovery which occurs on anaerobiosis has two components. One component represents a reversal of the changes which occur on membrane energization. The other component of the fluorescence change is insensitive to the uncoupler CCCP and resembles the behaviour of NPN with everted membrane vesicles. It is suggested that a portion of the fluorescence events seen with NPN involves a response of the probe to changes in the inner membrane.     

Characterization of specific fluorenylmethyloxycarbonyl-containing calmodulin adducts by spectroscopy and phosphodiesterase stimulation

A reagent (I, N⁴-(9-fluorenylmethyloxycarbonyl-4-amino-1-oxyl-4-succinimidyloxycarbonyl-2,2,6,6-tetramethylpiperidine)) that acylates calmodulin specifically at lysines 75 and 148 was recently described (Jackson and Puett, 1984). Chromatographic procedures are described that permit purification to apparent homogeneity of a 1 : 1 and a 2 : 1 adduct characterized by modification at just Lys 75 or at Lys 75 and Lys 148, respectively. These adducts are suitable for detailed characterization in an effort to provide information on calmodulin structure-function relationships. The adducts were incapable of, or exhibited low potency (e.g., 0.1% that of calmodulin) in, stimulating the activity of an activatable bovine brain cyclic nucleotide phosphodiesterase (3,5-cyclic AMP 5-nucleotidehydrolase, EC 3.1.4.17) preparation. Electron paramagnetic resonance (EPR) spectroscopy of the adducts yielded rotational correlation times of approximately 3–6 nsec, in agreement with the expected value for a hydrated protein of this molecular weight (5–7 nsec). Thus, the nitroxide reporter group appears to monitor closely the motion of the protein, and there is no evidence of a major conformational change in the derivative relative to calmodulin. Interestingly, removal of the fluorenylmethyloxycarbonyl portion from the 1 : 1 adduct to give a deprotected 1 : 1 adduct resulted in apparent greater mobility of the probe, since the rotational correlation coefficient was found to be 1 nsec. Circular dichroic spectra were obtained over the wavelength interval 200–250 nm on the two adducts and on the deprotected 1 : 1 adduct. These derivatives, like calmodulin, exhibited a Ca²⁺-mediated increase in helicity, and the spectra of the adducts in the presence of a chelating agent and in the presence of saturating Ca²⁺ were similar to those obtained for calmodulin. Thus, the adducts have secondary structures similar to the native protein. Proton nuclear magnetic resonance spectra were determined in the aromatic region (6–8 ppm) for the deprotected 1 : 1 adduct before and after reduction of the nitroxide with ascorbate. The nitroxide had little effect on the chemical shifts of the two tyrosines and the single histidine relative to calmodulin, although the histidine C4 resonance was markedly altered by the addition of ascorbate. In order to explore in greater detail the tertiary structure of the 1 : 1 adduct, a reagent similar to I, but not paramagnetic, was synthesized. This compound II, -N-(9-fluorenylmethyloxycarbonyl)alanine N-hydroxysuccinimide ester, like I, forms a 1 : 1 adduct at Lys 75 and a 2 : 1 adduct at Lys 75 and Lys 148. Proton NMR spectra of adducts with II were not complicated by the relaxation effects arising from adducts with I; thus more definitive assignments could be made to the upfield resonances, including the fluorene protons. Again, it was possible to conclude that adduct formation had no major effect on the tertiary structure of the protein as monitored by chemical shifts associated with various residues. We conclude that modification of just Lys 75, a residue in the long connecting helix of calmodulin, does not lead to major changes in protein conformation but does interfere with the ability of calmodulin to stimulate an activatable form of bovine brain cyclic nucleotide phosphodiesterase.     

The periplasmic nitrate reductase of Rhodobacter capsulatus; purification,characterisation and distinction from a single reductase for trimethylamine-N-oxide,dimethylsulphoxide and chlorate

The periplasmic dissimilatory nitrate reductase from Rhodobacter capsulatus N22DNAR⁺ has been purified. It comprises a single type of polypeptide chain with subunit molecular weight 90,000 and does not contain heme. Chlorate is not an alternative substrate. A molybdenum cofactor, of the pterin type found in both nitrate reductases and molybdoenzymes from various sources, is present in nitrate reductase from R. capsulatus at an approximate stoichiometry of 1 molecule per polypeptide chain. This is the first report of the occurrence of the cofactor in a periplasmic enzyme. Trimethylamine-N-oxide reductase activity was fractionated by ion exchange chromatography of periplasmic proteins. The fractionated material was active towards dimethylsulphoxide, chlorate and methionine sulphoxide, but not nitrate. A catalytic polypeptide of molecular weight 46,000 was identified by staining for trimethylamine-N-oxide reductase activity after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The same polypeptide also stained for dimethylsulphoxide reductase activity which indicates that trimethylamine-N-oxide and dimethylsulphoxide share a common reductase.Abbreviations DMSO dimethylsulphoxide - LDS lithium dodecyl sulphate - MVH reduced methylviologen - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - TMAO trimethylamine-N-oxide     

An 18 amino acid amphiphilic helix forms the membrane-anchoring domain of the Escherichia coli penicillin-binding protein 5

Small (10 residue) C-terminal deletions of PBP5 cause release of this Inner membrane protein into the periplasm, indicating disruption of the membrane binding domain. To define the extent of the membrane anchoring domain, oligonucleotide-directed mutagenesis was used to introduce both single amino acid changes and novel restriction sites into the DN A, allowing subsequent construction of precise internal deletions. The 10 C-terminal amino acid residues possess very weak membrane anchoring potential. By extending the sequence to 18 residues membrane binding equivalent to that of authentic PBP5 was achieved. A proline substitution in this region, breaking a potential α-helix, also disrupts the membrane binding domain. These results are discussed with respect to the amphi-philicity of the C-terminal sequence when arranged in an α-helix.     

Poly(gamma-glutamylcysteinyl)glycine Synthesis in Datura innoxia and Binding with Cadmium : Role in Cadmium Tolerance

The effects of Cd on poly(γ-glutamylcysteinyl)glycine [(γEC)_nG] biosynthesis and formation of (γEC)_nG:Cd complexes were measured in two cell lines of Datura innoxia with differing Cd tolerance. In addition, RNA synthesis, protein synthesis, and GSH concentrations were measured during a 48 hour exposure to Cd. Exposure to 250 micromolar CdCl₂ was toxic to the sensitive line, whereas the tolerant line survived and grew in its presence. Cd-sensitive cells synthesized the same amount of (γEC)_nG as tolerant cells during an initial 24 hour exposure to 250 micromolar CdCl₂. However, rates of (γEC)_nG:Cd complex formation differed between the two cell lines with the sensitive cells forming complexes later than tolerant cells. In addition, the complexes formed by sensitive cells were of lower molecular weight than those of tolerant cells and did not bind all of the cellular Cd. Pulse-labeling of cells with l-[³⁵S]cysteine resulted in equivalent rates of incorporation into the (γEC)_nG of both cell lines during the initial 24 hours after Cd. Rates of protein and RNA synthesis were similar for both cell lines during the initial 8 hours after Cd but thereafter declined rapidly in sensitive cells. This was reflected by a decline in viability of sensitive cells. The GSH content of both cell lines declined rapidly upon exposure to Cd but was higher in sensitive cells throughout the experiment. These results show that the biosynthetic pathway for (γEC)_nG synthesis in sensitive cells is operational and that relative overproduction of (γEC)_nG is not the mechanism of Cd-tolerance in a Cd-tolerant cell line of D. innoxia. Rapid formation of (γEC)_nG:Cd complexes that bind all of the cellular Cd within 24 hours appears to correlate with tolerance in these cells.     

Heterochromatin differentiation and phylogenetic relationship of the A genomes in diploid and polyploid wheats

Summary Heterochromatin differentiation, including band size, sites, and Giemsa staining intensity, was analyzed by the HKG (HCl-KOH-Giemsa) banding technique in the A genomes of 21 diploid (Triticum urartu, T. boeoticum and T. monococcum), 13 tetraploid (T. araraticum, T. timopheevi, T. dicoccoides and T. turgidum var. Dicoccon, Polonicum), and 7 cultivars of hexaploid (T. aestivum) wheats from different germplasm collections. Among wild and cultivated diploid taxa, heterochromatin was located mainly at centromeric regions, but the size and staining intensity were distinct and some accessions' genomes had interstitial and telomeric bands. Among wild and cultivated polyploid wheats, heterochromatin exhibited bifurcated differentiation. Heterochromatinization occurred in chromosomes 4A^t and 7A^t and in smaller amounts in 2A^t, 3A^t, 5A^t, and 6A^t within the genomes of the tetraploid Timopheevi group (T. araraticum, and T. timopheevi) and vice versa within those of the Emmer group (T. dicoccoides and T. turgidum). Similar divergence patterns occurred among chromosome 4A^a and 7A^a of cultivars of hexaploid wheat (T. aestivum). These dynamic processes could be related to geographic distribution and to natural and artifical selection. Comparison of the A genomes of diploid wheats with those of polyploid wheats shows that the A genomes in existing diploid wheats could not be the direct donors of those in polyploid wheats, but that the extant taxa of diploids and polyploids probably have a common origin and share a common A-genomelike ancestor.Contribution of the College of Agricultural Sciences, Texas Tech Univ. Journal No. T-4-233.