Tissue-specific silencing of a transgene in rice.

In a transgenic rice line, a beta-glucuronidase reporter gene under the control of the rice tungro bacilliform virus promoter became gradually methylated, and gene activity was lost concomitantly. Methylation was observed only in the homozygous offspring and was initially restricted to the promoter region and accompanied by loss of expression in the vascular bundle tissue only. This expression pattern was similar to that of a promoter with a deletion of a vascular bundle expression element. The gene activity could be reestablished by treatment with 5-azacytidine. Methylation per se did not inhibit the binding to the promoter region of protein factors which also bound to the unmethylated sequence. Instead, promoter methylation enabled the alternative binding of a protein with specificity for sequence and methylation. In further generations of homozygous offspring the methylation spread into the transcribed region and gene activity was completely repressed also in nonvascular cells. The results indicate that different stages are involved in DNA methylation-correlated gene inactivation, and that at least one of them may involve the attraction of a sequence and methylation-specific DNA-binding protein.

Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm.

Rice (Oryza sativa), a major staple food, is usually milled to remove the oil-rich aleurone layer that turns rancid upon storage, especially in tropical areas. The remaining edible part of rice grains, the endosperm, lacks several essential nutrients, such as provitamin A. Thus, predominant rice consumption promotes vitamin A deficiency, a serious public health problem in at least 26 countries, including highly populated areas of Asia, Africa, and Latin America. Recombinant DNA technology was used to improve its nutritional value in this respect. A combination of transgenes enabled biosynthesis of provitamin A in the endosperm.

Upstream and downstream sequence elements determine the specificity of the rice tungro bacilliform virus promoter and influence RNA production after transcription initiation.

The contribution of sequences upstream and downstream of the transcription start site to the strength and specificity of the promoter of rice tungro bacilliform virus was analysed in transgenic rice plants. The promoter is strongly stimulated by downstream sequences which include an intron and is active in all vascular and epidermal cells. Expression in the vascular tissue requires a promoter element located between -100 and -164 to which protein(s) from rice nuclear extracts bind. Elimination of this region leads to specificity for the epidermis. Due to the presence of a polyadenylation signal in the intron, short-stop RNA is produced from the promoter in addition to full-length primary transcript and its spliced derivatives. The ratio between short-stop RNA and full-length or spliced RNA is determined by upstream promoter sequences, suggesting the assembly of RNA polymerase complexes with different processivity on this promoter.

Software assessment under consideration of validation aspects: PPS and PMS systems.

Due to safety aspects, business risks and regulations of the FDA [FDA, 1983, Guide to Inspection of Computerized Systems in Drug Processing. USA] and the EU [EU-Leitfaden einer Guten Herstellpraxis for Arzneimittel, 1989, III/2244/87 Rev. 3. Jan. 1989], computer based systems used in the pharmaceutical production have to be validated. In this paper we assessed which modules of production planning and control (PPS) and production management systems (PMS) have to be validated using a risk analysis procedure. It could be shown that the necessity of the validation concerns, above all, the modules of production management systems. Furthermore, a software supplier questionnaire was developed according to the V-model of Royce [National Computing Centre, 1989. STARTS Publications, UK.]. The software supplier questionnaire was applied on four software products, which are mostly used in the pharmaceutical industry. Although these were developed according to a life cycle concept they fulfil the validation requirements, measured with the developed questionnaire, to a different degree. Especially the key element of the risk analysis was differently interpreted.

Human MxA protein confers resistance to Semliki Forest virus and inhibits the amplification of a Semliki Forest virus-based replicon in the absence of viral structural proteins.

Mx proteins form a small family of interferon (IFN)-induced GTPases with potent antiviral activity against various negative-strand RNA viruses. To examine the antiviral spectrum of human MxA in homologous cells, we stably transfected HEp-2 cells with a plasmid directing the expression of MxA cDNA. HEp-2 cells are permissive for many viruses and are unable to express endogenous MxA in response to IFN. Experimental infection with various RNA and DNA viruses revealed that MxA-expressing HEp-2 cells were protected not only against influenza virus and vesicular stomatitis virus (VSV) but also against Semliki Forest virus (SFV), a togavirus with a single-stranded RNA genome of positive polarity. In MxA-transfected cells, viral yields were reduced up to 1,700-fold, and the degree of inhibition correlated well with the expression level of MxA. Furthermore, expression of MxA prevented the accumulation of 49S RNA and 26S RNA, indicating that SFV was inhibited early in its replication cycle. Very similar results were obtained with MxA-transfected cells of the human monocytic cell line U937. The results demonstrate that the antiviral spectrum of MxA is not restricted to negative-strand RNA viruses but also includes SFV, which contains an RNA genome of positive polarity. To test whether MxA protein exerts its inhibitory activity against SFV in the absence of viral structural proteins, we took advantage of a recombinant vector based on the SFV replicon. The vector contains only the coding sequence for the viral nonstructural proteins and the bacterial LacZ gene, which was cloned in place of the viral structural genes. Upon transfection of vector-derived recombinant RNA, expression of the beta-galactosidase reporter gene was strongly reduced in the presence of MxA. This finding indicates that viral components other than the structural proteins are the target of MxA action.

Transgenic rice (Oryza sativa) endosperm expressing daffodil (Narcissus pseudonarcissus) phytoene synthase accumulates phytoene, a key intermediate of provitamin A biosynthesis.

Rice (Oryza sativa L.), the major food staple for more than two billion people, contains neither beta-carotene (provitamin A) nor C40 carotenoid precursors thereof in its endosperm. To improve the nutritional value of rice, genetic engineering was chosen as a means to introduce the ability to make beta-carotene into rice endosperm tissue. Investigation of the biochemical properties of immature rice endosperm using [14C]-labelled substrates revealed the presence of geranyl geranyl diphosphate, the C20 general isoprenoid precursor necessary for C40 carotenoid biosynthesis. Phytoene synthase, which condenses two molecules of geranyl geranyl diphosphate, is the first of four specific enzymes necessary for beta-carotene biosynthesis in plants. Therefore, the Japonica rice model variety Taipei 309 was transformed by microprojectile bombardment with a cDNA coding for phytoene synthase from daffodil (Narcissus pseudonarcissus) under the control of either a constitutive or an endosperm-specific promoter. In transgenic rice plants, the daffodil enzyme is active, as measured by the in vivo accumulation of phytoene in rice endosperm. Thus, it is demonstrated for the first time that it is in principle possible to engineer a critical step in provitamin A biosynthesis in a non-photosynthetic, carotenoid-lacking plant tissue. These results have important implications for long-term prospects of overcoming worldwide vitamin A deficiency.

Transgenic Indica rice breeding line IR58 expressing a synthetic cryIA(b) gene from Bacillus thuringiensis provides effective insect pest control.

The Indica rice breeding line IR58 was transformed by particle bombardment with a truncated version of a synthetic cryIA(b) gene from Bacillus thuringiensis. This gene is expressed under control of the CaMV 35S promoter and allows efficient production of the lepidopteran specific delta-endotoxin. R0, R1 and R2 generation plants displayed a significant insecticidal effect on several lepidopterous insect pests. Feeding studies showed mortality rates of up to 100% for two of the most destructive insect pests of rice in Asia, the yellow stem borer (Scirpophaga incertulas) and the striped stem borer (Chilo suppressalis), and feeding inhibition of the two leaffolder species Cnaphalocrocis medinalis and Marasmia patnalis. Introduction of stem borer resistance into the germplasm of an Indica rice breeding line now makes this agronomically important trait available for conventional rice breeding programs.

Fertile Indica rice plants regenerated from protoplasts isolated from scutellar tissue of immature embryos.

We report on the regeneration of fertile Indica rice (Oryza sativa L.) plants from protoplasts isolated from scutellar tissue of immature embryos. The average yields of protoplasts after purification ranged from 2.8 × 10(5) to 3.5 × 10(5) protoplasts per fifty embryos. Protoplasts developed rapidly to colonies when cultured in maltose containing medium using the nurse culture method. Upto 146 or 39 visible colonies per 10(6) protoplasts were obtained for the varieties Basmati 370 and IR43 respectively. Of two basal culture media compared, R2 medium containing 3 mg l(-1) kinetin, 1 mg l(-1) naphthalene acetic acid (NAA), 30 g l(-1) maltose and 3.0 g l(-1) agarose was found to be more effective in producing green plants. All scutellum protoplast-derived plants that were transferred to the greenhouse survived and were fertile.

Gene transfer by electroporation into intact scutellum cells of wheat embryos.

Gene transfer into intact cells was achieved by electroporating zygotic wheat embryos without any special pretreatment. Electroporation was tissue specific in so far as scutellum cells were found to be much more susceptible to gene transfer than other cell types of the embryo. The orientation of the embryos in the electroporation chamber also influenced the number of transformed scutellum cells; during electroporation, as in electrophoresis, the negatively charged plasmid DNA molecules seemed to move towards the positive electrode. Therefore, the embryos were arranged so that the scutella faced the negative electrode. The use of plasmids carrying either two chimeric anthocyanin regulatory genes or a chimeric gusA gene allowed clear identification of transformed cells in the scutellum. On some of the embryos, more than 100 transformed scutellum cells were found after electroporation with single electric pulses of 275 V/cm discharged from a 960-µF capacitor and with 100 µg DNA/ml electroporation buffer. Using the anthocyanin marker system, visibly transformed cells grew to produce red sectors.