The new pPZP Agrobacterium binary vectors are versatile, relatively small, stable and are fully sequenced. The vectors utilize the pTiT37 T-DNA border regions, the pBR322 bom site for mobilization from Escherichia coli to Agrobacterium, and the ColE1 and pVS1 plasmid origins for replication in E. coli and in Agrobacterium, respectively. Bacterial marker genes in the vectors confer resistance to chloramphenicol (pPZP100 series) or spectinomycin (pPZP200 series), allowing their use in Agrobacterium strains with different drug resistance markers. Plant marker genes in the binary vectors confer resistance to kanamycin or to gentamycin, and are adjacent to the left border (LB) of the transferred region. A lacZ alpha-peptide, with the pUC18 multiple cloning site (MCS), lies between the plant marker gene and the right border (RB). Since the RB is transferred first, drug resistance is obtained only if the passenger gene is present in the transgenic plants.

deleting the rpoB gene encoding the essential β subunit 1 Present address: University of Nebraska, Lincoln, of the tobacco PEP, we established the existence of a N209 Beadle Center, Lincoln, NE 68588-0664, USA second nuclear-encoded plastid RNA polymerase (NEP) in photosynthetic higher plants (Allison et al., 1996). Corresponding authorDeletion of rpoB yielded photosynthetically defective, pigment-deficient plants. An examination of ΔrpoB plants The plastid genome in photosynthetic higher plants encodes subunits of an Escherichia coli-like RNA polyrevealed proplastid-like structures containing low levels of mRNAs for the photosynthetic genes rbcL, psbA and merase (PEP) which initiates transcription from E.coli σ 70 -type promoters. We have previously established psbD due to the lack of PEP promoter activity. In wildtype tobacco leaves, the ribosomal RNA operon (rrn) is the existence of a second nuclear-encoded plastid RNA polymerase (NEP) in photosynthetic higher plants. We transcribed by PEP. Interestingly, in the ΔrpoB plants the rrn mRNA accumulated close to wild-type levels due to report here that many plastid genes and operons have at least one promoter each for PEP and NEP (Class II transcription by NEP acting at a downstream non-σ 70 -type promoter. The rRNA operon is the first plastid transcription unit). However, a subset of plastid genes, including photosystem I and II genes, are transcribed transcription unit for which a promoter for both PEP and NEP was identified. from PEP promoters only (Class I genes), while in some instances (e.g. accD) genes are transcribed excluWe report here that the rRNA operon is not unique, but represents a class of plastid transcription units which have sively by NEP (Class III genes). Sequence alignment identified a 10 nucleotide NEP promoter consensus at least one promoter each for PEP and NEP. These genes or operons have a potential for expression by either of around the transcription initiation site. Distinct NEP and PEP promoters reported here provide a general the two plastid RNA polymerases. Furthermore, some genes are transcribed by only one of the two RNA mechanism for group-specific gene expression through recognition by the two RNA polymerases.polymerases. We propose that transcription by NEP and PEP, through recognition of distinct promoters, is a general Keywords: Nicotiana tabacum/nuclear-encoded plastid RNA polymerase (NEP)/plastid-encoded RNA mechanism of group-specific gene regulation during chloroplast development. A tentative NEP promoter conpolymerase (PEP)/plastid gene expression/rpoB deficient mutant sensus is derived by the alignment of the transcription initiation sites.

Stable genetic transformation of the plastid genome is reported in a higher plant, Nicotiana tabacum. Plastid transformation was obtained after bombardment of leaves with tungsten particles coated with pZS148 plasmid DNA. Plasmid pZS148 (9.6 kilobases) contains a 3.7-kilobase plastid DNA fragment encoding the 16S rRNA. In the 16S rRNA-encoding DNA (rDNA) a spectinomycin resistance mutation is flanked on the 5' side by a streptomycin resistance mutation and on the 3' side by a Pst I site generated by ligating an oligonucleotide in the intergenic region. Transgenic lines were selected by spectinomycin resistance and distinguished from spontaneous mutants by the flanking, cotransformed streptomycin resistance and Pst I markers. Regenerated plants are homoplasmic for the spectinomycin resistance and the Pst I markers and heteroplasmic for the unselected streptomycin resistance trait. Transgenic plastid traits are transmitted to the seed progeny. The transgenic plastid genomes are products of a multistep process, involving DNA recombination, copy correction, and sorting out of plastid DNA copies.Transgenic plants are widely used to study nuclear gene function and regulation and to improve agronomically important crop plants (1, 2). Production of transgenic plants is achieved routinely by several alternative methods developed specifically for transformation of the nuclear genome of higher plants. Transgenic technology has not, however, been applied yet to genomes of plastids and mitochondria of higher plants.Introduction and stable integration of exogenous DNA have been reported recently in the plastid genome of a unicellular alga, Chlamydomonas reinhardtii (3-5). In this paper, we report stable genetic transformation of the plastid genome in a higher plant, Nicotiana tabacum. We propose the term transplastomic for the lines carrying a transgenic plastome. The transplastomic lines were selected by a nonlethal marker, resistance to the antibiotic spectinomycin that allows differentiation by color. On selective media the resistant clones are green and the sensitive clones are white (6). When cells are grown in a selective medium, plastids carrying the resistance genes are preferentially maintained (7). Selection, aided by an active system of recombination (8), was expected to facilitate the recovery of transplastomic clones.The antibiotic resistance markers were derived from N. tabacum SPC2, a line that exhibits a high level of resistance to spectinomycin and streptomycin due to mutations 278 base pairs (bp) apart in the 16S rRNA-encoding DNA (rDNA) (Z.S. and P.M., unpublished results). The 16S rDNA gene is in the repeated region and therefore is present in two copies per plastid genome. The SPC2 16S rDNA clone was further marked by a silent mutation resulting in a new restriction site to facilitate identification of transplastomic clones. Although the efficiency of transformation is low, the results reported in this paper demonstrate the requirements of plastid transformation in higher plants. MATERIALS AND MET...

Transgenic plants have become attractive systems for production of human therapeutic proteins because of the reduced risk of mammalian viral contaminants, the ability to do large scale-up at low cost, and the low maintenance requirements. Here we report a feasibility study for production of a human therapeutic protein through transplastomic transformation technology, which has the additional advantage of increased biological containment by apparent elimination of the transmission of transgenes through pollen. We show that chloroplasts can express a secretory protein, human somatotropin, in a soluble, biologically active, disulfide-bonded form. High concentrations of recombinant protein accumulation are observed (>7% total soluble protein), more than 300-fold higher than a similar gene expressed using a nuclear transgenic approach. The plastid-expressed somatotropin is nearly devoid of complex post-translational modifications, effectively increasing the amount of usable recombinant protein. We also describe approaches to obtain a somatotropin with a non-methionine N terminus, similar to the native human protein. The results indicate that chloroplasts are a highly efficient vehicle for the potential production of pharmaceutical proteins in plants.

SummaryWe describe here the development of a reproducible plastid transformation system for potato and regeneration of plants with uniformly transformed plastids. Two distinct tobacco-speci®c plastid vectors, pZS197 (Prrn/aadA/TpsbA) and pMON30125 (Prrn/GFP/ Trps16::PpsbA/aadA/TpsbA), designed for integration into the large single copy and inverted repeat regions of the plastid genome, respectively, were bombarded into leaf explants of potato line FL1607. A total of three transgenic lines were selected out of 46 plates bombarded with pZS197 and three transgenic lines out of 104 plates were obtained with pMON30125. Development of a high frequency leaf-based regeneration system, a stringent selection scheme and optimization of biolistic transformation protocol were critical for recovery of plastid transformants. Plastidexpressed green¯uorescent protein was used as a visual marker for identi®cation of plastid transformants at the early stage of selection and shoot regeneration. The establishment of a plastid transformation system in potato, which has several advantages over routinely used nuclear transformation, offers new possibilities for genetic improvement of this crop.