The glutamine synthetase-glutamate synthase system in Rhodopseudomonas sphaeroides

The phototrophic purple bacterium Rhodopseudomonas sphaeroides, strain 2R, can assimilate ammonium by means of glutamine synthetase and glutamate synthase. A higher activity of glutamine synthetase is displayed by cells grown in the medium with glutamate or in the atmosphere of molecular nitrogen. The activity of glutamate synthase also rises when cells grow in the atmosphere of N2. However, in contrast to glutamine synthetase, the activity of glutamate synthase does not decrease in the presence of considerable NH4+ amounts. The glutamine synthetase of R. sphaeroides is modified by adenylylation/deadenylylation. In the presence of nitrogenase in R. sphaeroides, the glutamine synthetase is found mainly in the deadenylylation state. Methionine sulfone, an inhibitor of glutamine synthetase, partly restores the activity of nitrogenase in the presence of ammonium, and prevents adenylylation of glutamine synthetase.     

Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation

Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants.     

Activity of the corn Spm transposon system in transgenic plants Orychophragmus violaceus (L.) O.E. Schulz obtained by both direct transfer of DNA to protoplasts and agrobacterial transformation of root explants

Transposon mediated insertional mutagenesis is one of the approaches for the unique gene cloning. A wild species of Cruciferae family Orychophragmus violaceus (L.) O.E. Schulz, which is of interest for practical breeding as a donor of improved plant oil, was an object of the investigation. Plasmid construction used in the experiments included selective NPT II gene, reported GUS gene serving as an excision marker, structural BAR gene located within the dSpm element and Spm transposase. The GUS gene of this plasmid had not his own promoter and became functional only after Spm-transposition. Transformed Orychophragmus violaceus (L.) O.E. Schulz. plants were obtained by direct mesophyll protoplast transformation as well as Agrobacterium tumefaciens-mediated root explant transformation. Gene transfer and the transposition event were confirmed by the GUS activity and the PCR analysis. Relative transformation efficiency using protoplasts was 5.8%.     

Obtaining and analysis of intergeneric somatic hybrids between Brassica napus and "albino" line of Orychophragmus violaceus

The Orychophragmus violaceus chlorophylldefective line of "albino" type has been obtained by spectinomycin treatment. Somatic hybridization between Orychophragmus violaceus and Brassica napus was performed by fusion of green mesophyll protoplasts of rape and callus protoplasts of the O. violaceus "albino" line. Near two hundred of regenerant plants were selected according to the regeneration type and ability to become green, and were determined as hybrids. Chloroplast DNA in selected hybrids was identical to rape chlDNA, which was confirmed by the PCR-RFLP analysis of plastid DNA fragments. Fragments of hybrid mitochondrial DNA analyzed by the PCR-RFLP analysis were identical to fragments of O. violaceus. The nuclear genome of the majority of hybrids was represented by the O. violaceus genome, which was demonstrated by analyses of isoenzymes, DNA telomeric sequences, ribosomal and satellite DNAs, and the RAPD analysis. The cytogenetic analysis of a number of lines has shown variability in the number of chromosomes in the obtained lines.     

Exogenous allogenic fragmented double-stranded DNA is internalized into human dendritic cells and enhances their allostimulatory activity

Exogenous allogenic DNA as nucleosome-free fragments reaches main cellular compartments (cytoplasm, nucleus) of human dendritic cells and deposits in the nuclear interchromosomal space without visibly changing in linear size. The presence of such allogenic fragmented DNA in medium in which human dendritic cells are cultured produces an enhancement of their allostimulatory activity. This enhancement is comparable to that produced by the standard maturation stimulus lipopolysaccharide Escherichia coli.     

Fatty acid composition variability of rapeseed oil: classical selection and biotechnology

The problems and achievements in the rapeseed Brassica napus L. var. oleifera breeding directed on the change of fatty acid composition in seed oil with the use of traditional and genetic engineering approaches are analyzed. It is noticed that the combination of biotechnological workings out and methods of classical breeding is the optimum for the further improvement of rapeseed oil composition.     

Stable expression of the promoterless bar gene in transformed rapeseed plants

Phosphinothricin-resistant plants of commercial summer rapeseed varieties (Brassica napus L. var. oleifera DC) were obtained by means of Agrobacterium tumefaciens-mediated transformation of leaf disks. The vector structures contained the promoterless coding sequence of the phosphinothricin acetyltransferase gene (bar), which is situated between two inverted lox sites (elements of the Cre/lox recombination systems of the PI phage) and the selective neomycinphosphotransferase II gene (nptII). The presence of the newly introduced genes in the genome of the obtained plants is confirmed by the polymerase chain reaction method. Stable and linked inheritance of these genes in the T₁ and T₂ generations is demonstrated.     

Establishment of transgenic lettuce plants producing potentially antihypertensive ShRNA

Development of RNAi-based therapeutics is a fast growing field of the pharmaceutical industry. Using plants for production of pharmaceutically valuable siRNAs may have significant advantages of costeffectiveness, scalability, and low risk of contamination with human pathogens. If edible plant species are genetically engineered to synthesize siRNAs, the costly stage of target product purification may be omitted. We describe the establishment of transgenic lettuce plants producing shRNA targeting delta isoform of protein kinase C (PKC-delta), an effective target for RNAi-based treatment of arterial hypertension. Transgenic lettuce plants were obtained by Agrobacterium-mediated transformation with genetic constructs harboring antiPKC and scrambled (control) shRNA genes. The presence of transgenes was proven by PCR analysis, and the accumulation of antiPKC shRNA was estimated using the RT-qPCR technique. Six transgenic lettuce lines showed varying levels of antiPKC shRNA expression with the highest value reaching 14 ± 9% of highly abundant endogenous lettuce micro RNA (miR156a), or 12.7 fmol/g dry weight. Plants carrying either antiPKC or scrambled shRNA genes flowered normally but did not produce seeds. The described transgenic lettuce plants accumulating antiPKC siRNA are the subject for animal testing and can be considered as raw material for the development of novel antihypertensive drugs.     

The role of intercellular interactions in the activation of T-lymphocyte colony precursors

The influence of the previous cultivation of human peripheral blood mononuclear cells in suspension with mitogen on the consequent PHA-stimulated response in suspension and agar cultures was studied. It has been established that proliferative response of mononuclear cells in suspension with mitogen is not changed, but the intensity of T-cell colony formation in two-layer agar systems in increased after preincubation mainly at the expense of increasing the number of type II colonies and clusters. It is concluded that the free cell-cell contacts are needed to activate T-lymphocyte colony precursors.