Cell genetic engineering ­ transmission genetics of plants.

It has been described achievements of cell and genetic engineering that led to formation of new genetics chapter ­ transmission genetics. It has been analyzed results and showed new opportunities in the field of transgenomic somatic hybrids and cybrid obtaining, production of transgenic plants with agronomic pharmaceutical application, development of transplastomic plants, accu-mulation of recombinant proteins by using the transient expression of foreign genes in plants.

Comparison of effectiveness of 5'-regulatory sequences in transplastomic tobacco chloroplasts.

The development of tools which ensure the desired level of transgene expression in plastids is a prerequisite for the effective utilization of these plant organelles for the deployment of bioactive proteins. High-level accumulation of target proteins is considered as a positive feature of transplastomic plants, but excessive accumulation of foreign proteins may have deleterious effects on host plants. On the other hand, expression at low levels can result in ineffective phenotypes. We compared the effectiveness of different 5'-regulatory sequences in driving the expression of a reporter gene, ß-glucuronidase (uidA), in tobacco chloroplasts. To achieve varying expression levels, we have chosen heterologous 5'-regulatory sequences which either differ significantly from their homologous counterparts or depend on specific nuclear encoded factors. The Medicago truncatula psbA promoter/5'-UTR supported the highest levels of protein accumulation, surpassing the other tested sequences by two to three orders of magnitude. The heterologous regulatory sequence of Phaseolus vulgaris rbcL gene was as efficient in tobacco chloroplasts as the corresponding homologous promoter/5'-UTR. The Arabidopsis thaliana ndhF promoter/5'-UTR supported as high reporter activity levels as the rbcL 5'-sequences, whereas the effectiveness of A. thaliana psbN promoter/5'-UTR was three fold lower. The characterized regulatory sequences can be utilized to establish transplastomic lines with desirable levels of target protein accumulation. The ability to control transgene expression should be useful for achieving appropriate levels of protein accumulation and thereby avoid their negative impacts on host plant physiology.

Early evolving joint degeneration by cartilage trauma is primarily mechanically controlled.

BACKGROUND: Mechanical and inflammatory processes add to osteoarthritis (OA). To what extent both processes contribute during the onset of OA after a cartilage trauma is unknown. This study evaluates whether local cartilage damage leads to focally confined or more generalized cartilage damage with synovial inflammation in the early development of joint tissue degeneration. METHODS: In nine goats, cartilage damage was surgically induced on the weight bearing area of exclusively the medial femoral condyle of the right knee joint. The other tibio-femoral compartments, lateral femoral condyle and lateral medial tibial plateau, were left untouched. The contralateral left knee joint of each animal served as an intra-animal control. Twenty weeks post-surgery changes in cartilage matrix integrity in each of the four compartments, medial and lateral synovial tissue inflammation, and synovial fluid IL-1ß and TNFα were evaluated. RESULTS: In the experimental medial femoral plateau, significant macroscopic, histologic, and biochemical cartilage damage was observed versus the contralateral control compartments. Also the articulating cartilage of the experimental medial tibial plateau was significantly more damaged. Whereas, no differences were seen between the lateral compartments of experimental and contralateral control joints. Synovial tissue inflammation was mild and only macroscopically (not histologically) significantly increased in the experimental medial compartments. Synovial fluid IL-1ß level was not different between experimental and contralateral control joints, and TNFα was overall beneath the detection limit. CONCLUSIONS: Local cartilage damage is a trigger for development of OA, which in early onset seems primarily mechanically driven. Early treatment of traumatic cartilage damage should take this mechanical component into consideration.

MULTIPLEX PCR ASSAY FOR DETECTION OF HUMAN SOMATOTROPIN AND INTERFERON ALPHA2b GENES IN PLANT MATERIAL.

Using transgenic plants as factories for production of physiologically active human proteins arouses special concern because occasional escape of such transgenes into environment may cause health problems. Creation of plant varieties producing pharmaceutically valuable proteins should be accompanied by development of detection methods suitable for controlling the transgene behavior. Here we describe a multiplex PCR protocol for revealing of two human genes (encoding growth hormone and interferon alpha2b) that have been successfully introduced into plant genomes. The primer pair designed for detection of human growth hormone coding sequence amplifies fragments of different size from the full-length gene in the human genome and the intronless coding sequence usually used for plant transformation. Application of this primer pair may be recommended for ruling out false positive results due to sample contamination with human DNA. Such a control may be useful also in PCR analysis during establishing of transgenic plants carrying genes of human origin.

Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression.

Human interferon alpha2b gene was transiently expressed in Nicotiana excelsior plants. Fusion with N. plumbaginifolia calreticulin signal peptide for improved apoplast targeting and carrying out the expression under optimized conditions resulted in maximal interferon activity of 3.2 x 10(3) IU/g fresh weight (FW) with an average of 2.1 +/- 0.8 x 10(3) IU/g FW. It proves that N. excelsior is a suitable host for Agrobacterium-mediated transient expression of genes encoding physiologically active human proteins. The transient expression conditions optimized for GFP marker protein were confirmed to be preferable for hIFN alpha2b.

Purification of recombinant GFP produced by Agrobacterum-mediated transient expression in Nicotiana excelsior.

Green fluorescent protein (GFP) is commonly used as a reporter protein in a wide range of biological experiments. The efficient protocol of Agrobacterium-mediated transient expression in Nicotiana excelsior was applied for quick preparative production of recombinant GFP. The protein purification scheme has been developed and included ammonium sulfate precipitation and Q-sepharose anion-exchange chromatography. It results in obtaining of a fraction with about 85% GFP homogeneity and the protein yield of about 75%.

Vitamin D deficiency as a risk factor for osteoporotic fractures.

The evidence on the association between vitamin D deficiency and fracture incidence is contradictory. Therefore, the objective of this study was to examine whether low serum 25-hydroxyvitamin D (25(OH)D) levels are associated with osteoporotic fractures. The study was conducted among 1311 community-dwelling older men and women of the Longitudinal Aging Study Amsterdam (LASA), an ongoing multidisciplinary cohort study. Serum 25(OH)D was determined using a competitive protein binding assay. Fractures were assessed during six years of follow-up. The data were analyzed using Cox proportional hazards model. In total, 11.3% of the persons had a serum 25(OH)D below 10 ng/ml, 48.4% had a value below 20 ng/ml, and 82.4% had a value below 30 ng/ml. Furthermore, 115 persons (8.5%) had one or more osteoporotic fractures. Different cut points of serum 25(OH)D were examined with a cut point of 12 ng/ml giving the best discrimination between persons with and without fractures (17.5% of the persons fell below this cut point). The lowest percentage of fractures (5.6%) was found above 30 ng/ml. Because an interaction effect with age was found (p=0.04), further analyses were conducted separately for persons aged 65-75 years (n=656) and for persons aged 75-89 years (n=664) at baseline. After adjustment for age, sex, season of blood collection, body mass index, number of chronic diseases, serum creatinine, cognition, smoking and alcohol use, serum 25(OH)D below or equal to 12 ng/ml was associated with an increased fracture risk in the youngest age group (HR=3.1; 95% CI: 1.4-6.9), but not in the oldest age group (HR=1.3; 95% CI: 0.7-2.2). For commonly used cut points of serum 25(OH)D (<10 ng/ml, 10-19.9 ng/ml, 20-29.9 ng/ml, > or =30 ng/ml), no statistically significant associations were found after adjustment for confounding. Serum 25(OH)D levels below or equal to 12 ng/ml were associated with an increased fracture risk in persons aged 65-75 years. The relatively low cut point of serum 25(OH)D in our population is possibly caused by high calcium intake in the Netherlands.

Comparison of several Nicotiana species as hosts for high-scale Agrobacterium-mediated transient expression.

Agrobacterium-mediated transient expression may be regarded as a promising method for inexpensive large-scale production of recombinant proteins. We optimized the protocol of transient expression in Nicotiana benthamiana and compared six Australian species of Nicotiana as hosts for transient expression. The transient expression of GFP under 35S CaMV promoter was observed in all species tested, although the GFP content in leaves of N. benthamiana, N. exigua, and N. excelsior was significantly higher (3.8, 3.7, and 2.0% TSP, respectively). Usage of viral-based expression system resulted in considerable increase of GFP accumulation in N. excelsior and N. benthamiana (63.5 and 16.2% TSP, respectively). We displayed that N. excelsior has the best characteristics in regard to biomass yield as well as GFP accumulation level for both types of the expression cassettes tested.

[Effect of lox-sites of the Cre lox recombination system on promoterless bar gene expression in transgenic plants].

We demonstrate that localization of lox site between the right border of T-DNA and promoterless bar gene (RB-lox-bar-) led to its highly efficient expression in transgenic plants of Nicotiana tabacum and N. africana. Plasmid vectors used in gene integration experiments contained neomycin phosphotransferase II (npt II) gene under nos promoter as well. Transgenic plants were selected according to their capacity to grow on the medium with kanamycin and then they were tested on the selective medium containing phosphinothricin. 80% of transgenic plants expressed bar gene at the level similar to that in plants transformed with the bar gene under widely used constitutive promoter. Transformation of plants with the plasmid vector containing only promoterless bar gene near T-DNA right border (RB-bar-) and with the vector containing lox site and promoterless bar gene in the middle of the construction (-lox-bar-) led to obtaining no more than 4.5% of transgenic plants resistant to phosphinothricin. PCR analyses confirmed both the absence of tandem repeats and of plasmid recombination resulting in transference of bar gene under promoter in plasmid vector. Nos-terminator situated between the lox site and the right border of T-DNA did not decrease bar gene expression.

Transgenic plants regenerated from hairy roots of Nicotiana benthamiana: a promising host for transient expression of foreign proteins.

Hairy root cultures of Nicotiana benthamiana have been obtained by co-cultivation of leaf explants with Agrobacterium rhizogenes strain A4 harboring a binary vector plasmid, and transgenic nature of the obtained cultures was confirmed by PCR analysis. Transgenic plants were regenerated from hairy roots. The biomass yield of transgenic plants grown in vitro was almost two-fold higher than those of wild-type N. benthamiana plants. They differed from untransformed plants by short internodes, reinforced stem, thick and wrinkled leaves and more developed root system. The level of Agrobacterium-mediated transient expression of green fluorescent protein (GFP) in the regenerated plants was similar to that of untransformed plants.

[In vitro regeneration of Nicotiana africana plants from explants of different type and mesophyll protoplasts]. / Regeneratsiia in vitro rastenii Nicotiana africana iz éksplantov raznogo tipa i mezofil'nykh protoplastov.

Methods of in vitro cultivation of an African endemic species Nicotiana africana (Solanaceae) possessing some valuable traits have been elaborated. Influence of different concentrations of auxin (alpha-naphtaleneacetic acid) and cytokinins (6-benzylaminopurine, zeatin, thidiazuron) on morphogenesis and plant regeneration has been analysed using leaves and internodes as explants. The optimum method of regeneration of N. africana shoots in leaf explants is cultivation in the presence of 0.1 mg/l NAA and 1 mg/l BA; in internode explants--cultivation on the medium containing 0.5 mg/l NAA and 1 mg/l zeatin. The use of thidiazuron was the most effective at the concentration of 0.4 mg/l with subsequent transfer of explants to hormone-free medium. Method of isolation and culture of mesophyll protoplasts enabling to regenerate N. africana plants during 3-4 months has been proposed.

[Genetic transformation of Nicotiana africana Merxm. with plasmids containing lox recombination]. / Geneticheskaia transformatsiia rastenii Nicotiana africana Merxm. plazmidami, soderzhashchimi saity recombinatsii lox.

An efficient genetic transformation method for african tobacco Nicotiana africana Merxm. has been established. African tobacco is a valuable source for cytoplasmic male sterility (CMS) and nuclear encoded resistance to potato virus Y (PVY). N. africana transgenic plants have been obtained using both Agrobacterium-mediated and direct transformation of leaf explants with gold particle bombardment using particle inflow gun. Plasmid vectors containing phosphinothricin resistance gene (bar gene) coding region without promoter and independent 35S promoter between lox sites (lox-bar-35S-lox) and nptII gene were used. Transgenic plants were selected according to growth capacity on the selective medium containing 50 mg/l kanamycin. PCR analyses of kanamycin-resistant plants confirmed the presence of nptII and bar genes in their genome. Agrobacterium-mediated transformation of root explants has proved to be the most efficient transformation method for N. africana.

[Analysis of nuclear and mitochondrial genomes of transplastomic Salpiglossis sinuata plants obtained by transfer of transformed plastids from N. tabacum (+S. sinuata) cybrid]. / Analiz iadernogo i mitokhondrial'nogo genomov u transplastomnykh rastenii Salpiglossis sinuata, poluchennykh putem perenosa transformirovannykh plastid ot tsibrida N. tabacum (+S. sinuata)

New model system of plastid transformation has been proposed using a wild representative of Solanaceae family--S. sinuata. Earlier obtained cybrid plants N. tabacum (+ S. sinuata) were used for transformation experiments by PEG treatment of protoplasts with aadA gene that confers resistance to spectinomycin. Transformed S. sinuata plastome was transferred from N. tabacum (+ S. sinuata) cybrid to S. sinuata wild type plants by somatic hybridization. Molecular analysis of nuclear and mitochondrial DNA has been performed.