Cloning and expression of a nuclear encoded plastid specific 33 kDa ribonucleoprotein gene (33RNP) from pea that is light stimulated.

We report the cloning and sequencing of both cDNA and genomic DNA of a 33 kDa chloroplast ribonucleoprotein (33RNP) from pea. The analysis of the predicted amino acid sequence of the cDNA clone revealed that the encoded protein contains two RNA binding domains, including the conserved consensus ribonucleoprotein sequences CS-RNP1 and CS-RNP2, on the C-terminus half and the presence of a putative transit peptide sequence in the N-terminus region. The phylogenetic and multiple sequence alignment analysis of pea chloroplast RNP along with RNPs reported from the other plant sources revealed that the pea 33RNP is very closely related to Nicotiana sylvestris 31RNP and 28RNP and also to 31RNP and 28RNP of Arabidopsis and spinach, respectively. The pea 33RNP was expressed in Escherichia coli and purified to homogeneity. The in vitro import of precursor protein into chloroplasts confirmed that the N-terminus putative transit peptide is a bona fide transit peptide and 33RNP is localized in the chloroplast. The nucleic acid-binding properties of the recombinant protein, as revealed by South-Western analysis, showed that 33RNP has higher binding affinity for poly (U) and oligo dT than for ssDNA and dsDNA. The steady state transcript level was higher in leaves than in roots and the expression of this gene is light stimulated. Sequence analysis of the genomic clone revealed that the gene contains four exons and three introns. We have also isolated and analyzed the 5' flanking region of the pea 33RNP gene.

Cloning and characterization of a cDNA encoding topoisomerase II in pea and analysis of its expression in relation to cell proliferation.

We have isolated and sequenced four overlapping cDNA clones to identify the full-length cDNA for topoisomerase II (PsTopII) from pea. Using degenerate primers, based on the conserved amino acid sequences of other eukaryotic type II topoisomerases, a 680 bp fragment was PCR-amplified with pea cDNA as template. This fragment was used as a probe to screen an oligo-dT-primed pea cDNA library. A partial cDNA clone was isolated that was truncated at the 3' end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of PsTopII is 4639 bp with an open reading frame of 4392 bp. The deduced amino acid sequence shows a strong homology to other eukaryotic topoisomerase II (topo II) at the N-terminus end. The topo II transcript was abundant in proliferative tissues. We also show that the level of topo II transcripts could be stimulated by exogenous application of growth factors that induced proliferation in vitro cultures. Light irradiation to etiolated tissue strongly stimulated the expression of topo II. These results suggest that topo II gene expression is up-regulated in response to light and hormones and correlates with cell proliferation. Besides, we have also isolated and analysed the 5'-flanking region of the pea TopII gene. This is first report on the isolation of a putative promoter for topoisomerase II from plants.

Isolation and characterisation of the cDNA encoding a glycosylated accessory protein of pea chloroplast DNA polymerase.

The cDNA encoding p43, a DNA binding protein from pea chloroplasts (ct) that binds to cognate DNA polymerase and stimulates the polymerase activity, has been cloned and characterised. The characteristic sequence motifs of hydroxyproline-rich glyco-proteins (HRGP) are present in the cDNA corres-ponding to the N-terminal domain of the mature p43. The protein was found to be highly O-arabinosylated. Chemically deglycosylated p43 (i.e. p29) retains its binding to both DNA and pea ct-DNA polymerase but fails to stimulate the DNA polymerase activity. The mature p43 is synthesised as a pre-p43 protein containing a 59 amino acid long transit peptide which undergoes stromal cleavage as evidenced from the post-translational in vitro import of the precursor protein into the isolated intact pea chloroplasts. Surprisingly, p43 is found only in pea chloroplasts. The unique features present in the cloned cDNA indicate that p43 is a novel member of the HRGP family of proteins. Besides p43, no other DNA-polymerase accessory protein with O-glycosylation has been reported yet.

Cloning, expression and characterization of a gene which encodes a topoisomerase I with positive supercoiling activity in pea.

We have isolated and sequenced the full length cDNA for topoisomerase I. Using degenerate primers, based on the conserved amino acid sequences of five eukaryotic topoisomerase I, a 386 bp fragment was PCR amplified using pea cDNA as template. This fragment was used as a probe to screen a pea cDNA library. Two partial cDNA clones were isolated which were truncated at the 5' end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of the gene was 3055 bp with an open reading frame of 2676 bp. The deduced structure of pea topoisomerase I contain 892 amino acids with a calculated molecular weight of 100 kDa and an estimated pI of 9.3. A comparison of the deduced amino acid sequences of the pea topo I with the other eukaryotic topoisomerases clearly suggested that they are all related. Pea topoisomerase I has been overexpressed in E. coli system and the recombinant topoisomerase purified to homogeneity. The purified protein relaxes both positive and negative supercoiled DNA in the absence of divalent cation Mg2+. In the presence of Mg2+ ions the purified enzyme introduces positive supercoils a unique property not reported in any other organism except in archaebacterial topoisomerase I. Polyclonal antibodies were raised against recombinant topoisomerase I and western blotting with sub-cellular fractions indicated the localization of this topoisomerase in pea nuclei.

Cloning and characterisation of a gene encoding an antiviral protein from Clerodendrum aculeatum L.

The Clerodendrum aculeatum-systemic resistance inducing (CA-SRI) protein, a 34 kDa basic protein, plays a key role in inducing strong systemic resistance in susceptible plants against various plant viruses [22]. We have cloned the cDNA encoding the CA-SRI from C. aculeatum leaves using antibodies raised against the purified protein and degenerate oligonucleotide probes derived from microsequencing of the CA-SRI protein. The full-length cDNA consisted of 1218 nucleotides with an open reading frame of 906 bp. The deduced amino acid sequence of CA-SRI protein showed varying homology (ranging from 11 to 54%) to the ribosome inactivating proteins (RIPs) from other plant species. CA-SRI inhibited in vitro protein synthesis both in rabbit reticulocyte lysate and wheat germ lysate but not in Escherichia coli in vitro translation system. The CA-SRI open reading frame was expressed in an E. coli expression vector and the purified recombinant protein inhibited protein synthesis in rabbit reticulocyte lysate. Southern blot analysis indicated that the CA-SRI gene may be present in low copy number.

A 43 kDa DNA binding protein from the pea chloroplast interacts with and stimulates the cognate DNA polymerase.

A DNA binding protein with DNA polymerase 'accessory activity' has been identified and purified to apparent homogeneity from pea chloroplasts. This protein consists of a single subunit of 43 kDa and binds to DNA regardless of its base sequence and topology. It increases cognate DNA polymerase-primase activity in a dose dependent manner. Using solid phase protein-protein interaction trapping and co-immunoprecipitation techniques, the purified protein was found to associate with the chloroplast DNA polymerase. The chloroplast DNA polymerase also binds directly to the radioiodinated 43 kDa protein. The specific interaction between 43 kDa protein and chloroplast DNA polymerase results in the synthesis of longer DNA chains. The 43 kDa protein, present abundantly in the pea chloroplast, appears to increase processivity of the chloroplast DNA polymerase and may play an important role in the replication of pea chloroplast DNA.

Purification and characterization of a DNA helicase from pea chloroplast that translocates in the 3'-to-5' direction.

An ATP-dependent DNA helicase has been purified to near homogeneity from pea chloroplasts. The enzyme is a homodimer of 68-kDa subunits. The purified enzyme shows DNA-dependent ATPase activity and is devoid of DNA polymerase, DNA topoisomerase, DNA ligase or nuclease activities. The enzyme requires Mg2+ or Mn2+ for its maximum activity. ATP is the most favoured cofactor for this enzyme while other NTP or dNTP are poorly utilized. Pea chloroplast DNA helicase can unwind a 17-bp duplex whether it has unpaired single-stranded tails at both the 5' end and 3' end, at the 5' end or at the 3' end only, or at neither end. However, it fails to act on a blunt-ended 17-bp duplex DNA. The enzyme moves unidirectionally from 3' to 5' along the bound strand. The unwinding activity is inhibited by the intercalating drugs nogalamycin and daunorubicine.

Characterisation and mode of in vitro replication of pea chloroplast OriA sequences.

A partially purified replicative system of pea chloroplast that replicates recombinant DNAs containing pea chloroplast origin sequences has been characterised. Polymerisation by this system is very fast and insensitive to chain terminators like dideoxynucleotides, arabinosylcytosine 5'-triphosphate, etc. Both strands of template DNA are synthesized and single-stranded DNA templates undergo more than one round of replication. When sequences of either of the two chloroplast origins of replication (OriA or OriB) are used as templates, the replicative intermediates are found to have sigma structures. Electron microscopic analysis of the sigma structures restricted with various enzymes reveals that the initiation site of in vitro replication maps near the displacement-loop regions where replication initiates also in vivo. Although the observed replication initiation in the OriA recombinant template is chloroplast-DNA-specific, the mode of replication is different from that observed in vivo with intact ctDNA. However, when the template DNA contains both the OriA and OriB sequences, the in vitro replication proceeds in the theta mode, the mode of replication usually observed in vivo.

Purification and characterization of a eukaryotic type 1 topoisomerase from pea chloroplast.

A 69-kDa protein with topoisomerase I activity has been homogeneously purified from the chloroplasts of pea leaves. The topoisomerase properties are detected in crude lysate of pea chloroplasts using the technique of transferring 32P radioactivity from the 32P-labeled DNA to the protein. The purified enzyme relaxes both positive and negative supercoils in topological steps of unity without requiring magnesium ions. The enzyme is sensitive to topoisomerase I-specific inhibitors like camptothecin and berenil, and unaffected by reagents like novobiocin and doxorubicin at the topoisomerase II-inhibitory dosage. In the presence of the enzyme, supercoiled DNA is nicked, and the 3'-phosphoryl end of the nick becomes covalently linked with the enzyme. A tyrosine residue of the enzyme is responsible for the covalent linkage. Rabbit antiserum raised against the 16-mer peptide spanning the active residues of human topoisomerase I recognizes the 69-kDa protein within the crude lysate of pea chloroplasts as does the antiserum to the purified 69-kDa protein. From the enzymatic characteristics, the protein has been classified as a eukaryotic type I topoisomerase.

Nascent transcript-binding protein of the pea chloroplast transcriptionally active chromosome.

This study describes the nascent RNA-binding protein of the pea chloroplast transcriptional complex. The protein has been identified by photoaffinity labelling of the transcriptionally active chromosome (TAC) which utilizes the endogenous plastid DNA as template. UV irradiation of lysed chloroplast or the isolated TAC under conditions optimized for transcription photocross-links nascent radiolabelled transcripts (up to 250 nucleotides in length) to a 48 kDa protein. The photoaffinity labelling of the transcript-binding protein is dependent on UV irradiation, is maximal after about 30 min of irradiation, and is completely dependent on transcriptional activity; no cross-linkage has been observed with pre-synthesized RNA. Cross-linkage is influenced by salts and inhibitors in accordance with their effects on transcription. The photoconjugate is composed of protein and RNA moieties, and can be hydrolysed by several proteases. However, the cross-linked transcript is protected from nucleases until the protein is removed. Manganese enhances photoaffinity labelling of the transcript-binding protein, and this is paralleled by an increase in total transcriptional activity of TAC. This protein was isolated by 2-dimensional polyacrylamide gel electrophoresis and the sequence of 15 amino acid residues at the amino terminus was determined. The nascent transcript-binding protein appears to be involved in the transcription of all three classes of chloroplast genes. We also found a polypeptide of identical molecular weight to get cross-linked to nascent transcripts in chloroplasts isolated from other legumes such as Cicer arietenum, Vigna radiata and Phaseolus vulgaris, and monocots like Zea mays, Oryza sativa and Pennisetum americanum.

Characterization of the pea chloroplast DNA OriA region.

One of the two origins of replication in pea chloroplast DNA (oriA) maps in the rRNA spacer region downstream of the 16S rRNA gene, and further characterization of this origin is presented here. End-labeling of nascent DNA strands from in vivo replicating ctDNA was used to generate probes for Southern hybridization. Hybridization data identified the same region that was previously mapped to contain D-loops by electron microscopy. Subclones of the oriA region were tested for their ability to support in vitro DNA replication using a partially purified pea ctDNA replication system. Two-dimensional agarose gel electrophoresis identified replication intermediates for clones from the region just downstream of the 16S rRNA gene, with a 450-bp SacI-EcoRI clone showing the strongest activity. The experiments presented in this paper identify the 940 base pair region in the rRNA spacer between the 3' end of the 16S rRNA gene and the EcoRI site as containing oriA. Previous studies by electron microscopy localized the D-loop in the spacer region just to the right of the BamHI site, but the experiments presented here show that sequences to the left of the BamHI site are required for replication initiation from oriA. DNA sequence analysis of this region of pea ctDNA shows the presence of characteristic elements of DNA replication origins, including several direct and inverted repeat sequences, an A + T rich region, and dnaA-like binding sites, most of which are unique to the pea ctDNA oriA region when compared with published rRNA spacer sequences from other chloroplast genomes.

Purification and characterization of ribonucleoproteins from pea chloroplasts.

RNA-binding proteins are known to mediate the post-transcriptional regulation of genes in many organisms. Recently they have been found to be important in the expression of plastid genes. We have purified a group of three single-stranded nucleic-acid-specific acidic proteins (33, 30 and 28 kDa) from chloroplast extracts of pea (Pisum sativum L.), using single-stranded DNA affinity chromatography. All of them have acidic amino termini but the amino acid sequences are unique to each polypeptide, with partial similarities to the recently reported ribonucleoproteins from tobacco chloroplasts. The pea proteins are also antigenically distinct, as shown by Western blot analysis using polyclonal antisera for purified proteins. Further, from their large nucleic-acid-binding domains and the polynucleotide substrate affinities, they are predicted to belong to a family of pea plastid ribonucleoproteins. In vivo radiolabeling of proteins in the presence of translational inhibitors as well as in vitro translation of leaf tissue RNA suggest that these proteins are encoded in the nucleus. Antibody cross-reactivity experiments reveal that their genes are conserved during plastid evolution.

Two distinct transcriptional activities of pea (Pisum sativum) chloroplasts share immunochemically related functional polypeptides.

An RNA polymerase activity has been purified from pea (Pisum sativum) chloroplast extracts with a distinct transcriptional specificity for a chloroplast messenger gene. This activity (ms-RNA pol) differs from the pea RNA polymerase preparation reported by Sun, Shapiro, Wu & Tewari [(1986) Plant Mol. Biol. 6, 429-439], which specifically transcribes only the rRNA gene (rb-RNA pol). The specificity of transcription has been assessed by the synthesis in vitro of discrete transcripts of predicted sizes using cloned promoter regions of the chloroplast psbA and 16 S rRNA genes. The ms-RNA pol preparation, with polypeptides ranging in apparent molecular mass from 22 to 180 kDa, correctly initiates transcription from recombinant plasmids containing the psbA promoter and does not support 16 S rRNA promoter-directed transcription. The two activities differ also in their response to Mn2+ ions. To investigate whether the two transcriptional activities share common functional polypeptides, monoclonal antibodies were developed against the rb-RNA pol preparation. Three clones were selected on the basis of their ability to inhibit transcription in vitro of the 16 S rRNA gene by rb-RNA pol. The antibodies from these clones independently recognized three polypeptides with molecular masses of 27, 90 and 95 kDa on immunoblots. Antibodies cross-reacting with the 90 kDa polypeptide completely eliminated the specific retardation of an end-labelled 16 S rRNA promoter fragment in a mobility-shift assay, whereas the antibodies against the 95 kDa polypeptide resulted in the formation of a ternary complex (enzyme-DNA-antibody). The antibodies cross-reacting with the 27 kDa polypeptide, however, did not alter the mobility of the retarded DNA-enzyme complex on the gel. These antibodies also inhibited transcription in vitro of the psbA gene by ms-RNA pol and recognized polypeptides of identical molecular masses in the ms-RNA pol. These results show that the three polypeptides are functional components of the chloroplast transcriptional complex and appear to be involved in the transcription of both rRNA and mRNA genes. Transcriptional specificity is probably conferred by ancillary transcription factor(s) which remain to be identified.

Identification of the template binding polypeptide in the pea chloroplast transcriptional complex.

We have identified the template-binding polypeptide in the pea chloroplast transcriptional complex by photoaffinity labelling. This polypeptide has an apparent molecular weight of about 150 kDa and binds to both, chloroplast ribosomal (16S rRNA) and messenger (psbA) promoters. The 16S rRNA and psbA promoters were amplified from chloroplast DNA by the polymerase chain reaction and labelled with a photoactive analogue of TTP, 5-bromodeoxy UTP, as well as with alpha-32P-dCTP. Using the filter-binding assay, the conditions for binding of the RNA polymerase complex to chloroplast promoters were optimized. The polypeptide directly interacting with the template was photo-crosslinked to it and resolved by denaturing gel electrophoresis. The photoaffinity labelling of the 150 kDa polypeptide was dependent on photoactivation by UV irradiation, and the presence of chloroplast promoters. Competition experiments showed that the protein formed a strong interaction with the plastid promoters which could not be displaced by lambda-phage DNA or synthetic polynucleotides. The photo-crosslinked and nuclease-treated promoter-polypeptide complex was resistant to further digestion with DNase and RNase, but could be hydrolyzed by Proteinase K. Binding of the promoters by the 150 kDa polypeptide could not be surpressed by transcription inhibitors like rifampicin and alpha-amanitin. However, heparin (0.001%) inhibited the formation of the enzyme-promoter complex, and interfered with the photoaffinity labelling of the 150 kDa polypeptide. The extent of photoaffinity labelling of 150 kDa polypeptide exhibits some degree of correlation to total transcriptional activity under various salt concentrations. The results demonstrate that the 150 kDa polypeptide is a functional template binding polypeptide of the pea chloroplast transcription complex.

Photoaffinity labelling of the pea chloroplast transcriptional complex by nascent RNA in vitro.

We have used photoaffinity labelling to examine the chloroplast RNA polymerase components which come into contact with nascent transcripts during the in vitro transcription of plastid DNA. The transcripts were synthesized in the presence of a photoactive analogue (4-thio UTP) and alpha-32P-ATP, using enriched pea chloroplast RNA polymerase preparation and a recombinant plasmid containing the plastid 16S rRNA promoter. Brief irradiation of the transcriptional complex crosslinked the photoactive nascent RNA to proximal proteins. Labelling of the transcriptional complex was dependent on 4-thio UTP and template DNA. Two polypeptides of 51 and 54 kDa were consistently crosslinked to the nascent transcripts; about 60% of the total radioactivity of the crosslinked RNA was associated with these polypeptides. In some experiments, two additional polypeptides of 38 and 75 kDa were also found to be associated with about 13% and 17% of the total crosslinked RNA radioactivity, respectively. The UV-crosslinked transcriptional complexes were stable to either DNase or S1 nuclease hydrolysis but partially sensitive to RNase T1. Insensitivity of the complex to hydrolysis with RNase H suggested that the nascent transcripts were not crosslinked to the template. The complexes could also be hydrolysed by proteinase K and thermolysin. No crosslinkage was observed when labelled RNA molecules containing 4-thio UMP residues were added after synthesis to the polymerase preparation. This suggested that the method identified only those polypeptides which came into close contact with the transcript during its synthesis. Antibodies raised against the RNA-protein complex confirmed the presence of the polypeptides in the chloroplast RNA polymerase preparation on Western blots. Preincubation of these antibodies with the chloroplast RNA polymerase inhibited plastid DNA transcription. These data showed that the transcript-binding polypeptides were functional components of the chloroplast transcriptional complex.

Pea chloroplast DNA primase: characterization and role in initiation of replication.

A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19 + 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of 3H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115-120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19 + 2.1. Primers synthesized using M13mp19 + 2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.

Highly purified pea chloroplast RNA polymerase transcribes both rRNA and mRNA genes.

Pea chloroplast RNA polymerase has been obtained with about 2000-fold purification using DEAE-cellulose and phosphocellulose chromatography. The purified enzyme contained ten prominent polypeptides of 150, 130, 115, 110, 95, 85, 75, 48, 44 and 39 kDa and four other minor polypeptides of 90, 34, 32 and 27 kDa. Purification of this enzyme using chloroplast 16S rDNA promoter affinity column chromatography also yielded an enzyme with similar polypeptides. Purified polyclonal antibodies against the purified chloroplast RNA polymerase were found to recognize most of the polypeptides of the enzyme in Western blot experiments. Primary mobility shift of the 16S rRNA gene and ribulose-1,5-bisphosphate carboxylase large subunit (rbc-L) gene promoters observed with the chloroplast RNA polymerase was abolished by these antibodies. The specific in vitro transcription of these rRNA and mRNA genes was also inhibited by these antibodies. The transcription of the rRNA and mRNA genes was also abolished by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase. The chloroplast RNA polymerase was found to bind specifically to the chloroplast 16S rRNA gene promoter region as visualized in electron microscopy. The presence of the polypeptides of 130, 110, 75-95 and 48 kDa in the DNA-enzyme complex was confirmed by a novel approach using immunogold labeling with the respective antibodies. The polypeptides of this purified RNA polymerase were found to be localized in chloroplasts by an indirect immunofluorescence.

Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors.

In vitro analysis of the pea chloroplast 16S rRNA gene promoter.

A cloned pea chloroplast 16S rRNA gene promoter has been characterized in detail by use of a homologous in vitro transcription system that contains a highly purified chloroplast RNA polymerase. The in vivo and in vitro 16S rRNA transcriptional start site has been identified to be a T on the plus strand, 158 bases upstream of the mature 5' end of the gene. BAL 31 deletions of the 16S rRNA leader region demonstrated that the bases between -66 to +30 relative to the transcriptional start site (+1) are necessary for specific 16S transcription. Disruption of canonical TTGACA or TATAAT elements within this region caused complete transcriptional inactivation and prevented protein binding. The topological requirement for 16S transcription was examined by using a construct that synthesized a transcript from the 16S promoter and released it from a pea plastid putative terminator sequence. This minigene was relaxed in vitro with a topoisomerase I from pea chloroplast. It was shown that the 16S promoter was most active when the minigene plasmid was supercoiled.

Localization of replication origins in pea chloroplast DNA.

The locations of the two replication origins in pea chloroplast DNA (ctDNA) have been mapped by electron microscopic analysis of restriction digests of supercoiled ctDNA cross-linked with trioxalen. Both origins of replication, identified as displacement loops (D-loops), were present in the 44-kilobase-pair (kbp) SalI A fragment. The first D-loop was located at 9.0 kbp from the closest SalI restriction site. The average size of this D-loop was about 0.7 kbp. The second D-loop started 14.2 kbp in from the same restriction site and ended at about 15.5 kbp, giving it a size of about 1.3 kbp. The orientation of these two D-loops on the restriction map of pea ctDNA was determined by analyzing SmaI, PstI, and SalI-SmaI restriction digests of pea ctDNA. One D-loop has been mapped in the spacer region between the 16S and 23S rRNA genes. The second D-loop was located downstream of the 23S rRNA gene. Denaturation mapping of recombinants pCP 12-7 and pCB 1-12, which contain both D-loops, confirmed the location of the D-loops in the restriction map of pea ctDNA. Denaturation-mapping studies also showed that the two D-loops had different base compositions; the one closest to a SalI restriction site denatured readily compared with the other D-loop. The recombinants pCP 12-7 and pCB 1-12 were found to be highly active in DNA synthesis when used as templates in a partially purified replication system from pea chloroplasts. Analysis of in vitro-synthesized DNA with either of these recombinants showed that full-length template DNA was synthesized. Recombinants from other regions of the pea chloroplast genome showed no significant DNA synthesis activity in vitro.