Stable transformation of petunia plastids

Plastid transformation results in stably expressed foreign genes, which for most Angiosperms are largely excluded from sperm cells, thereby greatly reducing the risk of foreign gene spread through pollen. Prior to this work, fertile plastid transformants were restricted to tobacco, tomato and Lesquerella . Application of plastid engineering in the important floriculture industry requires the development of stable plastid transformation in a major ornamental plant species such as Petunia hybrida. Here we describe the successful isolation of fertile and stable plastid transformants in a commercial cultivar of P. hybrida (var. Pink Wave). Plastid targeting regions from tobacco were used to integrate aad A and gusA between the acc D and rbc L genes of P. hybrida plastid DNA following particle bombardment of leaves. For three spectinomycin and streptomycin resistant lines, DNA blot analysis confirmed transgene integration into plastid DNA and homoplasmy. Maternal inheritance and homoplasmy resulted in 100 transmission of spectinomycin resistance to progeny after selfing. Plastid transformants expressed the gusA gene uniformly within leaves and to comparable levels in all three lines. Insertion of trait genes in place of gusA coding sequences enables immediate applications of our plastid transformation vector. Establishment of plastid transformation in P. hybrida facilitates a safe and reliable use of this important ornamental plant for research and plant biotechnology.These two authors contributed equally to this work.     

Comparative genomics of Pneumocystis carinii with other protists: implications for life style

Three protistan genomes were analyzed for differential genetic traits that may be associated with biological adaptations to their unique life styles. The microsporidian, Encephalitozoon cuniculi, an obligate intracellular parasite; the ascomycetes, Pneumocystis carinii, considered an opportunistic pathogen; and Saccharomyces cerevisiae, a model organism exhibiting a free-living life style, were used in comparisons of genomic architecture, reproductive strategies, and metabolic capacity predicted by the presence of signature genes. Genome size, gene number, and metabolic function decreased as the organisms became more dependent on their hosts. In contrast, gene density and the percentage of genes dedicated to cell growth and division were substantially increased in the genome of E. cuniculi. The obligate life style was associated with reductions in gene number, genome size, and reduced metabolic capacity while the free-living life style was coincident with gene duplications and duplication of large portions of the genome. The genomic characteristics and metabolic capacity of P. carinii were usually intermediate between those of the other two protistan genomes, but unique characteristics such as the presence of a single rDNA locus may indicate that these organisms could be in the process of becoming more host dependent.     

Monomers for Oligonucleotide Synthesis with Linkers Carrying Reactive Residues: II. The Synthesis of Phosphoamidites on the Basis of Uridine and Cytosine and Containing a Linker with Methoxyoxalamide Groups in Position 2′

A convenient preparative synthesis of 2-amino-2-deoxyuridine was developed. Starting from 2-amino-2-deoxyuridine and 2-amino-2-deoxycytidine, monomers for the phosphoamidite oligonucleotide synthesis were obtained that carry a linker with methoxyoxalamide groups in position 2.     

Effect of molecular structure on thermodynamic properties of carbohydrates. A calorimetric study of aqueous di- and oligosaccharides at subzero temperatures

For aqueous solutions of di- and oligosaccharides thermodynamic properties have been investigated at subzero temperatures using differential scanning calorimetry. The amount of unfrozen water observed is found to increase linearly with the glass transition temperatures of anhydrous carbohydrates. Furthermore, the amount of unfrozen water shows a linear relationship with known solution properties of aqueous carbohydrates, such as partial molar compressibility and heat of solution. The different effectiveness among various di- and oligosaccharides to avoid ice formation is associated with the combination of constitutive monosaccharides and attendant molecular structure features including the position and type of the glycosidic linkage between the constituent units. More unfrozen water is induced in the presence of a carbohydrate having a poorer compatibility with the three-dimensional hydrogen-bond network of water. A series of these results obtained imply that there is a common key of carbohydrate stereochemistry governing several different thermodynamic amounts of a given system involving carbohydrates. In this context, a modified stereospecific-hydration model can be used to interpret the present results in terms of stereochemical effects of carbohydrates.     

Conformational changes in chemically modified Escherichia coli thioredoxin monitored by H/D exchange and electrospray ionization mass spectrometry

Hydrogen/deuterium (H/D) exchange in combination with electrospray ionization mass spectrometry and near-ultraviolet (UV) circular dichroism (CD) was used to study the conformational properties and thermal unfolding of Escherichia coli thioredoxin and its Cys32-alkylated derivatives in 1% acetic acid (pH 2.7). Thermal unfolding of oxidized (Oxi) and reduced (Red) -thioredoxin (TRX) and Cys-32-ethylglutathionyl (GS-ethyl-TRX) and Cys-32-ethylcysteinyl (Cys-ethyl-TRX), which are derivatives of Red-TRX, follow apparent EX1 kinetics as charge-state envelopes, H/D mass spectral exchange profiles, and near-UV CD appear to support a two-state folding/unfolding model. Minor mass peaks in the H/D exchange profiles and nonsuperimposable MS- and CD-derived melting curves, however, suggest the participation of unfolding intermediates leading to the conclusion that the two-state model is an oversimplification of the process. The relative stabilities as measured by melting temperatures by both CD and mass spectral charge states are, Oxi-TRX, GS-ethyl-TRX, Cys-ethyl-TRX, and Red-TRX. The introduction of the Cys-32-ethylglutathionyl group provides extra stabilization that results from additional hydrogen bonding interactions between the ethylglutathionyl group and the protein. Near-UV CD data show that the local environment near the active site is perturbed to almost an identical degree regardless of whether alkylation at Cys-32 is by the ethylglutathionyl group, or the smaller, nonhydrogen-bonding ethylcysteinyl group. Mass spectral data, however, indicate a tighter structure for GS-ethyl-TRX.     

Analysis of genetic effects of major genes and polygenes on quantitative traits I. Genetic model for diploid plants and animals

A genetic model was proposed to simultaneously investigate genetic effects of both polygenes and several single genes for quantitative traits of diploid plants and animals. Mixed linear model approaches were employed for statistical analysis. Based on two mating designs, a full diallel cross and a modified diallel cross including F₂, Monte Carlo simulations were conducted to evaluate the unbiasedness and efficiency of the estimation of generalized least squares (GLS) and ordinary least squares (OLS) for fixed effects and of minimum norm quadratic unbiased estimation (MINQUE) and Henderson III for variance components. Estimates of MINQUE (1) were unbiased and efficient in both reduced and full genetic models. Henderson III could have a large bias when used to analyze the full genetic model. Simulation results also showed that GLS and OLS were good methods to estimate fixed effects in the genetic models. Data on Drosophila melanogaster from Gilbert were used as a worked example to demonstrate the parameter estimation. Received: 11 November 2000 / Accepted: 2 May 2001     

Stabilization of trypsin by chemical modification with β-cyclodextrin monoaldehyde

The monoaldehyde derivative of -cyclodextrin was attached to trypsin via reductive alkylation with NaBH₄. The thermostability was enhanced from 49.5 °C to 60 °C for modified trypsin. The activation free energy of thermal inactivation at 50 °C was increased by 3.2 kJ mol^–1. The conjugated enzyme retained 100% of its initial activity after 3 h incubation at pH 9.     

Bollworm responses to release of genetically modified Helicoverpa armigera nucleopolyhedroviruses in cotton

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) has been developed as a commercial biopesticide to control the cotton bollworm, H. armigera, in China. The major limitation to a broader application of this virus has been the relative long time to incapacitate the target insect. Two HaSNPV recombinants with improved insecticidal properties were released in bollworm-infested cotton. One recombinant (HaCXW1) lacked the ecdysteroid UDP-glucosyltransferase (egt) gene and in another recombinant (HaCXW2), an insect-selective scorpion toxin (AaIT) gene replaced the egt gene. In a cotton field situation H. armigera larvae treated with either HaCXW1 or HaCXW2 were killed faster than larvae in HaSNPV-wt treated plots. Second instar H. armigera larvae, which were collected from HaCXW1 and HaCXW2 treated plots and further reared on artificial diet, showed reduced ST(50) values of 15.3 and 26.3%, respectively, as compared to larvae collected from HaSNPV-wt treated plots. The reduction in consumed leaf area of field collected larvae infected with HaCXW1 and HaCXW2 was approximated 50 and 63%, respectively, as compared to HaSNPV-wt infected larvae at 108 h after treatment. These results suggest that in a cotton field situation the recombinants will be more effective control agents of the cotton bollworm than wild-type HaSNPV.     

Functionalization of the Oligonucleotides Containing an Internucleotide Phosphoramidate Bond

A new method for functionalization of oligonucleotides by addition of aminoalkyl derivatives to the intermolecular phosphorus atom of the oligonucleotide N3"–P5" phosphoramidate bond in the presence of triphenylphosphine, 4-dimethylaminopyridine, and 2,2"-dipyridyl disulfide was suggested. The reaction proceeded with both low-molecular alkylamines (1,6-diaminohexane or N,N-dimethyl-1,3-diaminopropane) and a minor groove binder containing an aminoalkyl group.