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.

No abstract

The plastid genome in higher plants encodes subunits of an Escherichia coli‐like RNA polymerase which initiates transcription of plastid genes from sequences resembling E.coli sigma70‐type promoters. By deleting the gene for the essential beta subunit of the tobacco E.coli‐like RNA polymerase, we have established the existence of a second plastid transcription system which does not utilize E.coli‐like promoters. In contrast to the E.coli‐like RNA polymerase, the novel transcription machinery preferentially transcribes genetic system genes rather than photosynthetic genes. Although the mutant plants are photosynthetically defective, transcription by this polymerase is sufficient for plastid maintenance and plant development.

Chloroplast genomes defied the laws of Mendelian inheritance at the dawn of plant genetics, and continue to defy the mainstream approach to biotechnology, leading the field in an environmentally friendly direction. Recent success in engineering the chloroplast genome for resistance to herbicides, insects, disease and drought, and for production of biopharmaceuticals, has opened the door to a new era in biotechnology. The successful engineering of tomato chromoplasts for high-level transgene expression in fruits, coupled to hyper-expression of vaccine antigens, and the use of plant-derived antibiotic-free selectable markers, augur well for oral delivery of edible vaccines and biopharmaceuticals that are currently beyond the reach of those who need them most.Chloroplast transformation is an environmentally friendly approach to plant genetic engineering that minimizes out-crossing of transgenes to related weeds or crops [1,2] and reduces the potential toxicity of transgenic pollen to non-target insects [3]. Because the plastid genome is highly polyploid, transformation of chloroplasts permits the introduction of thousands of copies of foreign genes per plant cell, and generates extraordinarily high levels of foreign protein [3]. Chloroplast transformation vectors use two targeting sequences that flank the foreign genes and insert them, through homologous recombination, at a precise, predetermined location in the organelle genome (Fig. 1). This results in uniform transgene expression among transgenic lines and eliminates the 'position effect' often observed in nuclear transgenic plants. Gene silencing, frequently observed in nuclear transgenic plants, has not been observed in genetically engineered chloroplasts. The ability to express foreign proteins at high levels in chloroplasts and chromoplasts, and to engineer foreign genes without the use of antibiotic resistant genes [4,5],make this compartment ideal for the development of edible vaccines [6]. Moreover, the ability of chloroplasts to form disulfide bonds and to fold human proteins has opened the door to high-level production of biopharmaceuticals in plants [7]. Furthermore, foreign proteins observed to be toxic in the cytosol are non-toxic when accumulated within transgenic chloroplasts [6,8]. Chloroplast and nuclear genetic engineering are compared in Table 1.

Using DNA encoding the largest subunit of Drosophila melanogaster RNA polymerase II, we isolated the homologous hamster RP021 gene. Nucleotide sequencing of both the hamster and D. melanogaster RP021 DNAs confirmed that the RP021 polypeptides of these two species, like the Saccharomyces cerevisiae RP021 polypeptide, contain both an N-terminal region homologous to the Escherichia coli RNA Biochemical and genetic approaches are being used to study the molecular mechanisms which regulate mRNA transcription in eucaryotes. Through such approaches, regulatory cis-acting DNA elements have been identified and protein factors that recognize these specific sequences have been described. These DNA-binding transcription factors must directly or indirectly affect the activity of RNA polymerase II. To address this aspect of transcriptional regulation, a more thorough understanding of this enzyme itself may be required. Toward this end, we and others have used mutations that increase the a-amanitin resistance of RNA polymerase II to select, identify, or characterize other RNA polymerase II mutations (11,15,20). An a-amanitin resistance mutation in RNA polymerase II has also been used for the chromosomal mapping (11) and molecular cloning (25) of DNA encoding the largest subunit (10) of Drosophila melanogaster RNA polymerase II.Previous studies in this laboratory showed that the RNA polymerase II DNA from the D. melanogaster RP021 (also called RpII215 in reference 2) locus hybridized to mammalian DNA. Using DNA-mediated gene transfer, we identified cross-hybridizing restriction fragments in DNA from Chinese hamster ovary (CHO), Syrian hamster, and human cells that encode at least part of the mammalian RNA polymerase II polypeptide (16). Subsequently this D. melanogaster DNA was used to isolate the analogous polymerase II genes from Saccharomyces cerevisiae (17), human (4), and mouse (5) cells. Nucleotide-sequencing studies of the yeast genes RP021 and RP031 (la), which encode the largest subunit of RNA polymerase II (la,17) and RNA polymerase III (21), respectively, and a similar analysis of a portion of the D. melanogaster RP021 DNA (2) established that the eucaryotic RNA polymerase polypeptides are remarkably similar in * Corresponding author. their primary structure to the analogous subunit of the Escherichia coli RNA polymerase, ,B'. In addition, the largest subunit of yeast (la) and mouse (5) RNA polymerase II was found to contain a C-terminal extension. This domain consists of an evolutionarily conserved, tandemly repeated heptapeptide sequence with the consensus sequence TyrSer-Pro-Thr-Ser-Pro-Ser.In the present study we report the isolation of DNA encoding the Chinese hamster RNA polymerase II RP021 polypeptide and the nucleotide sequences of the 3' portion of both this hamster gene and the D. melanogaster gene. Like the RP021 polypeptide of S. cerevisiae