The circular chloroplast DNAs from spinach, lettuce, and corn plants have been examined by electron microscopy and shown to contain a large sequence repeated one time in reverse polarity. The inverted sequence in spinach and lettuce chloroplast DNA has been found to be 24,400 base pairs long. The inverted sequence in the corn chloroplast DNA is 22,500 base pairs long. Denaturation mapping studies have shown that the structure of the inverted sequence is highly conserved in these three plants. Pea chloroplast DNA does not contain an inverted repeat. All of the circular dimers of pea chloroplast DNA are found to be in a head-to-tail conformation. Circular dimers of spinach and lettuce were also found to have head-to-tail conformation. However, approximately 70-80% of the circular dimers in preparations of lettuce and spinach chloroplast DNA were found to be in a head-to-head conformation. We propose that the head-to-head circular dimers are formed by a recombination event between two circular monomers in the inverted sequence.The chloroplast DNA (ctDNA) from higher plants exists as covalently closed circular DNA molecules (1-6). The ctDNA from the alga Euglena gracilis has also been found to be circular (7). The ctDNA molecules from different species of higher plants have significantly different sizes and range from a Mr of 85.4 X 106 for corn ctDNA (2) to a Mr of 97.2 X 106 for lettuce ctDNA (2, 3). Euglena ctDNA has a Mr of approximately 90 X 106 (2, 8). The excellent agreement between the molecular weights of ctDNAs from higher plants determined by kinetics complexity and electron microscopy suggests that the sequence of a circular ctDNA molecule represents the entire informational content of the ctDNA (ref. 3; unpublished results). This suggestion has been further confirmed by denaturation mapping studies on pea ctDNA which have shown that all of the pea ctDNA molecules exhibit the same denaturation pattern (6).We have been studying the structural relationships among the ctDNAs from different species of higher plants in order to obtain information on the divergence of ctDNAs through evolution. In this communication the ctDNA molecules from four different higher plants (pea, spinach, lettuce, and corn) have been examined for the presence of sequences that are repeated in reverse. Such sequences renature spontaneously after denaturation (9) and have been observed in a large variety of DNAs ranging from bacterial plasmids (9) to eukaryotic chromosomal DNA (10). The data obtained have shown that the ctDNAs from lettuce, spinach, and corn plants contain a similar sequence, comprising approximately 16% of their monomer length, that is repeated one time in reverse. In pea ctDNA this sequence has not been found in inverted repeats. The structure of ctDNA circular dimers has been also investigated and the results suggest that they might be formed by a recombination mechanism.MATERIALS AND METHODS DNA. Covalently closed circular ctDNA from corn (Zea mays), spinach (Spinacia oleracea), lettuce (Lactuca sativa), a...

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The chloroplast DNA (ctDNA) from pea and corn plants contains both Cairns type and rolling circle replicative intermediates. Denaturation mapping studies with pea ctDNA molecules have shown that the rolling circles initiate replication at or near the site where the Cairns replicative intermediates terminate replication. These results suggest that the rolling circles are initiated by a Cairns round of replication. A model for the replication of the chloroplast DNA is based on these results.

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, pACpl8, 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. Organellespecific 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.

The mitochondrial DNA from pea leaves exists in a circular conformation. 25% of the circular molecules exist as supercoils, and 10% of the molecules are dimers. The molecular weight of mitochondrial DNA is about 66 to 70 X 106 by electron microscopy, and 74 X 106 from its renaturation kinetics. No evidence for inter-and intramolecular heterogeneity is found.The mitochondrial (mt-) DNA from animal tissues exists in the form of closed circular duplex molecules of 10 X 106 molecular weight (1). Luck and Reich (2, 3) reported the isolation of linear molecules of molecular weight 13 X 106 from Neurospora mitochondria, and calculated that the molecular weight of mt-DNA might be about 66 X 106 from its renaturation kinetics. Recent studies (4) have shown that mt-DNA from Neurospora can be obtained in a linear structure 26 jum in length. Similarly, mt-DNA from slime mold Physarium has been isolated in linear forms, with molecular weights ranging from 20 to 30 X 106 (5). Extensive studies in osmotically shocked preparations of yeast have demonstrated the existence of mt-DNA in circular form (6). However, all attempts to isolate these circular molecules -were unsuccessful. The molecular weight of yeast mt-DNA was calculated to be 50 X 106 (6). Mt-DNA from protozoa, Tetrahymena, and Paramecium, are linear structures of 17.6 jim in length (7). Mt-DNA from higher plants has always been isolated in linear molecules with mean lengths ranging from 10 to 20 ,um (7,8). We have studied mt-DNA from pea leaves; it has a molecular weight of 74 X 106 from its renaturation rate. The DNA released from osmotically shocked mitochondria, as well as that isolated by deproteinization, has a circular form with an average contour length of 30 jum under experimental conditions in which replicative form (RF) II DNA from bacteriophage OX-174 has a length of 1.45 Mum. This is the largest circular mt-DNA isolated to date. MATERIALS AND METHODSI8olation of Mitochondrial DNA. 12-to 15-day-old pea leaves were homogenized with 4 volumes of buffer containing 0.3 M mannitol-0.05 M Tris HCl (pH 8.0)-3 mM EDTA-0.1% bovine-serum albumin-1 mM 2-mercaptoethanol.Homogenates were filtered through cheese cloth and centrifuged at 1020 X g for 15 min; the supernatant was then centrifuged at 12,000 X g for 20 min. The mitochondrial fraction (contaminated with broken nuclei and chloroplasts) was suspended in the buffer (200 ml/kg of leaves) and centrifuged twice at 1020 X g for 15 min; the final mitochondrial pellet was obtained after centrifugation at 12,000 X g for 20 min. This pellet was suspended in the buffer (40 ml/kg of leaves) and treated with 50 MLg/ml of DNase for 1 hr at 4°.All subsequent operations to obtain DNA free from RNA and protein were those reported elsewhere (9).Isolation of Mitochondrial DNA for Electron Microscopy. 100 g of leaves were chopped with razor blades in 250 ml of buffer as reported (10). The mitochondrial pellet obtained as described above was suspended in 10 ml of buffer, treated with DNase, and washed -three times by suspe...

A DNA polymerase has been purified >3,000-fold from the chloroplasts of pea plants by chromatography on DEAE-cellulose, phosphocellulose, single-stranded DNA-agarose, and sedimentation in a glycerol gradient. Electrophoretic analysis on polyacrylamide gels in the presence of sodium dodecyl sulfate indicates that the final fraction contained a single discernible protein band of 90,000 daltons. Gel filtration on Sephacryl S-200 and glycerol gradient sedimentation under nondenaturing conditions demonstrate that the chloroplast DNA polymerase has a native molecular mass of approximately 87,000 daltons. The purified polymerase lacks any associated nuclease activity. The enzyme activity is inhibited by N-ethylmaleimide (74% at 1.0 mM) and ethidium bromide (90% at 0.23 mM) and is resistant to aphidicolin. The purified enzyme is totally dependent on the presence of added DNA, has an absolute requirement for Mg2+ (12 mM optimal), is stimulated by K+ (120 mM optimal), and requires all four deoxynucleoside triphosphates for maximum activity. Native DNA which has been degraded to a limited extent with DNase I is the most efficient template.The chloroplast genome in higher plants exists as circular DNA molecules of 130-150 kilobase pairs (kbp). The chloroplast DNAs (ctDNAs) from higher plants have been found to be largely homogeneous in sequence organization as shown by renaturation kinetics, electron microscopy, denaturation mapping, and restriction endonuclease digestions (1-3). The replication of ctDNA has been studied by analyzing the replicating ctDNA molecules with electron microscopy (4,5

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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)