Showing papers by "Pal Maliga published in 1999"

Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants.

Abstract: Plastid transformation in higher plants is accomplished through a gradual process, during which all the 300-10,000 plastid genome copies are uniformly altered Antibiotic resistance genes incorporated in the plastid genome facilitate maintenance of transplastomes during this process Given the high number of plastid genome copies in a cell, transformation unavoidably yields chimeric tissues, which requires the identification of transplastomic cells in order to regenerate plants In the chimeric tissue, however, antibiotic resistance is not cell autonomous: transplastomic and wild-type sectors both have a resistant phenotype because of phenotypic masking by the transgenic cells We report a system of marker genes for plastid transformation, termed FLARE-S, which is obtained by translationally fusing aminoglycoside 3"-adenyltransferase with the Aequorea victoria green fluorescent protein 3"-adenyltransferase (FLARE-S) confers resistance to both spectinomycin and streptomycin The utility of FLARE-S is shown by tracking segregation of individual transformed and wild-type plastids in tobacco and rice plants after bombardment with FLARE-S vector DNA and selection for spectinomycin and streptomycin resistance, respectively This method facilitates the extension of plastid transformation to nongreen plastids in embryogenic cells of cereal crops

In vitro characterization of the tobacco rpoB promoter reveals a core sequence motif conserved between phage-type plastid and plant mitochondrial promoters

Abstract: We report here the in vitro characterization of PrpoB-345, the tobacco rpoB promoter recognized by NEP, the phage-type plastid RNA polymerase. Transcription extracts were prepared from mutant tobacco plants lacking PEP, the Escherichia coli-like plastid-encoded RNA polymerase. Systematic dissection of a approximately 1 kb fragment determined that the rpoB promoter is contained in a 15-nucleotide segment (-14 to +1) upstream of the transcription initiation site (+1). Point mutations at every nucleotide reduced transcription, except at the -5 position which was neutral. Critical for rpoB promoter function was a CRT-motif (CAT or CGT) at -8 to -6 (transcription <30%), defining it as the promoter core. The core CAT sequence is also present in the maize rpoB promoter, which is faithfully recognized by tobacco extracts. Alignment of NEP promoters identified a CATA or TATA (=YATA) sequence at the rpoB core position, also present in plant mitochondrial promoters. Furthermore, NEP and the phage T7 RNA polymerase exhibit similar sensitivity to inhibitors of transcription. These data indicate that the nuclear RpoZ gene, identified by sequence conservation with mitochondrial RNA polymerases, encodes the NEP catalytic subunit.

Plastome Engineering of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Tobacco to Form a Sunflower Large Subunit and Tobacco Small Subunit Hybrid

Abstract: Targeted gene replacement in plastids was used to explore whether the rbcL gene that codes for the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase, the key enzyme of photosynthetic CO2 fixation, might be replaced with altered forms of the gene. Tobacco (Nicotiana tabacum) plants were transformed with plastid DNA that contained the rbcL gene from either sunflower (Helianthus annuus) or the cyanobacterium Synechococcus PCC6301, along with a selectable marker. Three stable lines of transformants were regenerated that had altered rbcL genes. Those containing the rbcL gene for cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase produced mRNA but no large subunit protein or enzyme activity. Those tobacco plants expressing the sunflower large subunit synthesized a catalytically active hybrid form of the enzyme composed of sunflower large subunits and tobacco small subunits. A third line expressed a chimeric sunflower/tobacco large subunit arising from homologous recombination within the rbcL gene that had properties similar to the hybrid enzyme. This study demonstrated the feasibility of using a binary system in which different forms of the rbcL gene are constructed in a bacterial host and then introduced into a vector for homologous recombination in transformed chloroplasts to produce an active, chimeric enzyme in vivo.

Translation control elements for high-level protein expression in the plastids of higher plants and methods of use thereof

Abstract: DNA constructs containing translational control elements are provided. These 5' regulatory segments facilitate high level expression of transgenes introduced into the plastids of higher plants.

Transplastomic Technology for Safer and Better Transgenic Crops

Abstract: Pal Maliga received his Ph.D. degree in 1972 from the University of Szeged, Hungary. His early research at the Biological Research Center in Szeged focused on cell biology approaches to manipulate the plastid and mitochondrial genomes of higher plants. In 1988, he was appointed Professor of Genetics at Rutgers, The State University of New Jersey. Since then his research group at the Waksman Institute has developed the technology for plastid transformation in higher plants using tobacco as a model system. They currently use plastid transformation to characterize the plastid transcription machinery and to understand the rules of mRNA translation, RNA editing and production of recombinant proteins in plastids. In addition, they are directing their efforts to extend the technology of plastid transformation to Arabidopsis and rice. Transplastomic Technology for Safer and Better Transgenic Crops

A Transgenic Approach to Characterize the Plastid Transcription Machinery in Higher Plants

Abstract: The plastid genes of higher plants are transcribed by two RNA polymerases: the plastid-encoded plastid RNA polymerase or PEP and the nuclear-encoded plastid RNA polymerase or NEP. Below is a brief summary of what we know about the two plastid RNA polymerases, highlighting recent data from this laboratory. Most of the data were obtained using transgenic approaches, facilitated by the development of technology for plastid transformation in tobacco [1, 2].

Engineering the Plastid Genome: Problems and Potential

Abstract: The plastid genome of higher plants is a 120-kb to 160-kb double-stranded DNA present in 1,900 to 50,000 copies per leaf cell. To obtain genetically stable transplastomic lines every one of the plastid genome copies (ptDNA) should be uniformly altered in a plant. Transformation is accomplished through the following steps: (i) introduction of the transforming DNA, encoding antibiotic resistance, by the biolistic process or PEG treatment; (ii) integration of the transforming DNA by two homologous recombination events and (iii) elimination of wild-type genome copies during repeated cell divisions on a selective medium. As integration of foreign DNA always occurs by homologous recombination, plastid transformation vectors contain segments of the plastid genome to target insertions to specific locations. The plastid vectors also contain a marker for selection. Useful, non-selectable genes are cloned next to selectable marker genes, with which they are introduced into the plastid genome. Homology-directed genome manipulations have included introduction of point-mutations and deletion of targeted genes.