Circular Chloroplast Research Articles

Arrangement of the ribosomal RNA genes in chloroplast DNA of Leguminosae

The circular chloroplast DNA from three species of plants in the taxonomic family Leguminosae were examined using electron microscopic techniques and restriction endonuclease digestion. Chloroplast DNAs from chickpea (Cicer arietinum), mung bean (Vigna radiata), and soy bean (Glycine max) were found to range in size from 119–151 kilobase pairs by contour length measurements. Sizes of the chloroplast DNAs have been further confirmed using different restriction endonucleases. Two of the chloroplast DNAs examined, soy bean and mung bean, contain a region approximately 15.9–18% of their monomer length that is repeated in reverse polarity. This repeated region separates a small unique region that ranges in size from 18.75–20.4 kilobase pairs and a large unique region that ranges in size from 73.4–85 kbp. This feature was not found in the chloroplast DNA of chickpea. R-loop hybridizations performed using chloroplast ribosomal RNAs demonstrate that the two ribosomal gene sets of the mung been and soy bean are arranged in inverted orientation within this repeated region. In contrast, the chickpea chloroplast DNA posesses a single ribosomal RNA gene set in the circular molecule. In all three chloroplast DNAs examined, the genes encoding the chloroplast 23S and 16S ribosomal RNA genes are separated by a spacer region which ranges in size from 2.2 to 2.48 kbp.

Visualization of a Spinach Plastid Transcriptionally Active DNA-Protein Complex in a Highly Condensed Structure

A transcriptionally active DNA-protein complex isolated from spinach Spinacia oleracea plastids is visualized by electron microscopy in different conditions. This structure, after glutaraldehyde fixation, is highly condensed. DNA is supertwisted with proteins bound to it producing a beaded substructure. When glutaraldehyde fixation is omitted this structure is less condensed and DNA fibrils come out from a proteinous central body. The DNA-protein complex can be separated into two populations by CsCl centrifugation: one with a buoyant density of 1.570 grams per cubic centimeter and the other of 1.610 grams per cubic centimeter. By visualization of these two populations, it is concluded that proteins are either firmly bound to DNA in the central body, or more loosely bound to the DNA fibrils. These latter proteins could play a role in enzymic functions and/or in the supercoiling of DNA.The DNA from the DNA-protein complex possesses all fragments that belong to pure circular chloroplast DNA hydrolyzed by two restriction enzymes: Bam HI and Eco RI. Some molecules observed in a supercondensed form with a beaded substructure probably contain entire chloroplast DNA molecules.A hydrolysis test with microccocal nuclease gives no indication of the presence of ;nucleosome-like' structures. Thirty-six polypeptides with molecular weights ranging from 12,000 to 180,000 are present in the complex, and seven of them are highly soluble in 0.4 n H(2)SO(4); their molecular weights range from 14,000 to 46,000 as shown by two-dimensional gel electrophoresis.No linolenic acid can be detected in the preparation, indicating the absence of chloroplast membranes.

Rearrangements in the chloroplast genomes of mung bean and pea

We have mapped all the cleavage sites for the restriction endonucleases BstEII, Kpn I, Pst I, Pvu II, Sac I, Sal I, Sma I, and Xho I on the circular chloroplast chromosomes from mung bean and pea. The mung bean chloroplast genome measures 150 kilobase pairs (kb) in length; it includes two identical sequences of 23 kb that contain the ribosomal genes and are arranged as an inverted repeat separated by single-copy regions of 21 and 83 kb. The pea chloroplast genome is only 120 kb in size, has only one set of ribosomal genes, and does not possess any detectable repeated sequences. The mung bean inverted repeat structure is common to all other nonleguminous higher plant chloroplast genomes studied, whereas the pea structure has been found only in the closely related legume Vicia faba. We conclude from these data that loss of one copy of the inverted repeat sequence has occurred only rarely during the evolution of the Angiosperms, and in the case of the legumes after the divergence of the mung bean line from the pea-Vicia line. We present hybridization data indicating that rearrangements that change the linear order of homologous sequences within the chloroplast genome have been quite frequent during the course of legume evolution.

Construction of a SalI/ PstI restriction map of spinach chloroplast dna using low-gelling-temperature-agarose electrophoresis

The restriction endonucleases SalI and PstI cleave circular chloroplast DNA of spinach ( Spinacia oleracea) into 12 and 10 fragments, respectively. The sum of the fragment sizes in each of the series is equivalent to the contour length of the molecule (about 95 Md). A physical map was constructed by sequential digestions using low-gelling-temperature agarose to avoid the necessity of extracting the fragments from the gel. The circular DNA molecule of spinach chloroplasts consists of two identical sequences (each about 15 Md) arranged as an inverted repeat separated by two single-copy regions of different sizes (about 52 and 13 Md).

Inverted repeats in chloroplast DNA from higher plants

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 confirmation. 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.

Anatomy of the chloroplast ribosomal DNA of chlamydomonas reinhardii

Two ribosomal DNA (rDNA) regions exist in the circular chloroplast genome of C. reinhardii. They are located at widely distant sites on the map in an inverted orientation. The chloroplast rDNA has been cloned and a fine structure map has been established. The small chloroplast ribosomal subunit contains 16S RNA, while the large subunit contains the four distinct RNA species 23, 7, 5 and 3S rRNA. The location of the rRNA genes has been determined by electron microscopy and by hybridization of the purified rRNAs to various rDNA restriction fragments. The gene order in the direction of transcription is 16, 7, 3, 23 and 5S. A spacer (Sp) of 1.68 ± 0.13 kb separates the 16S and the 7S RNA genes, and a very small spacer of 0.16 ± 0.04 kb separates the 7S and 23S RNA genes. The 5, 23, 3 and 7S RNAs are transcribed on the same strand. A surprising result is the interruption of all chloroplast 23S rRNA genes by a 940 base pair (bp) intervening sequence at a site 0.27 ± 0.04 kb distant from the 5′ end of the 23S RNA coding strand. This interruption has been observed both in cloned and uncloned chloroplast rDNA restriction fragments. When single-stranded chloroplast rDNA is mounted for electron microscopy, distinctive features of secondary structure which flank the rRNA gene sequences can be seen.

Leaf Anatomy of Petunia in Relation to Pollution Damage1

Abstract Anatomical observations were made on 2 petunia cultivars of Petunia hybrida Vilm., one ozone and sulfur dioxide susceptible and the other moderately tolerant with leaf cross and paradermal sections made from collections taken 4 hours to 30 days following fumigation. Disintegration of the circular chloroplast bodies into one homogenous mass was the first visible symptom of internal cell damage. Initially, O3 affected chloroplasts in palisade cells while SO2 affected chloroplasts in spongy cells. Plasmolysis, aggregation of cytoplasmic contents, and disintegration of nuclei occurred. Damage from SO2 proceeded more rapidly and was more extensive than that from O3; all cell components were destroyed over larger areas. Foliar sprays of succinic acid-2,2-dimethyl-hydrazide (daminozide) or benomyl soil drenches were applied 1 week before fumigation. Microscopic observations indicated that mesophyll tissue in both daminozide and benomyl treatments resembled to some degree the compact tissue typical of young, insensitive leaves.

Zea mays chloroplast ribosomal RNA genes are part of a 22,000 base pair inverted repeat.

Zea mays chloroplast rDNA exists in two identical units. Each unit contains one sequence for the 16, 23 and 5S rRNAs in the order given. The 16 and 23S sequences in each unit are separated by a 2100 base pair (bp) spacer. The DNA sequence for 5S RNA is closely linked to that for the 23S RNA. Within the above unit, the three RNAs are transcribed from a single DNA strand. The two rDNA units on the circular chloroplast DNA molecule are separated from each other by 18,500 bp in one direction and by 106,100 bp in the other direction. The two rDNA units have an inverted orientation with respect to each other. Each rDNA unit is part of a 22,000 bp sequence which is repeated with inverted orientation.

Restriction endonuclease map of Euglena gracilis chloroplast DNA

A physical map of the Euglena gracilis chloroplast genome has been constructed, based on cleavage sites of Euglena gracilis chloroplast DNA treated with bacterial restriction endonucleases. Covalently close, circular chloroplast DNA is cleaved by restriction endonuclease SalI into three fragments and by restriction endonuclease BamHI into six fragments. These nine cleavage sites have been ordered by fragment molecular weight analysis, double digestions, partial digestions, and by digestion studies of isolated DNA fragments. A fragment pattern of the products of EcoRI restriction endonuclease digestion of Euglena chloroplast DNA is also described. One of these fragments has been located on the cleavage site map.

Circular molecules in the β-satellite DNA of Chlamydomonas reinhardii

The β-satellite DNA (chloroplast DNA) of Chlamydomonas reinhardii has been shown to contain a homogeneous population of circular molecules of about 62 μm contour length. Evidence is presented that this molecule is a monomer and that the β-satellite is a homogeneous DNA species. A second class of circular molecules of as yet unknown intracellular origin, having a circumference of about 27 μm, has also been observed. Our data demonstrate that the sizes of circular chloroplast DNA vary considerably even within the Chlorophyta.

Physical studies on the size and structure of the covalently closed circular chloroplast DNA from higher plants

The size and structure of the covalently closed circular chloroplast DNAs (ctDNA) from pea, lettuce, and spinach plants, have been studied by analytical ultracentrifugation. The values of s o 20,w,Na + of the native and denatured forms of the open and closed circular DNAs from these plants have been determined. The absolute molecular weight of purified closed circular pea ctDNA monomers has been determined by buoyant equilibrium sedimentation to be 89.1 (S.D. ± 0.7) · 10 6. The value of the s o 20,w,Na + of open circular pea ctDNA and its molecular weight, in conjunction with corresponding values for other sizes of circular DNA, has been used to derive an empirical relationship between s o 20,w,Na + and molecular weight for open circular DNAs. Using this relationship, the molecular weights of lettuce and spinach ctDNAs have been determined to be 98.2 (S.D. ± 1.5) · 10 6 and 97.2 (S.D. ± 1.5) · 10 6, respectively. At pH values 12.7 and 13, closed circular lettuce and pea ctDNAs have been found to exist as mixtures of reversibly and irreversibly denatured closed circular DNAs.

Presence of displacement loops in the covalently closed circular chloroplast deoxyribonucleic acid from higher plants.

Chloroplast DNAs (ctDNA) from pea and corn plants were examined in the electron microscope for the presence of replicative intermediates. Pea and corn ctDNAs were each found to contain two displacement loops (D-loops). The D-loops were 820 (+/- 90) base pairs long in pea ctDNA and 860 (+/- 125) base pairs long in corn ctDNA. In each ctDNA, the two D-loops were located at positions that were 7100 +/- 240) base pairs apart. The displacing strands of the two D-loops were located on opposite strands of the parental DNA molecule and they were seen to expand toward each other. The D-loops in the ctDNA from pea and corn exhibited branch migration and thus were easily distinguished from the denatured regions that were also present in these closed circular ctDNAs. In addition, the positions of the D-loops were found to be distinct from the positions of the denaturation loops (Den-loops). The Den-loops were also shown to be located at AT-rich regions in these ctDNA molecules. D-loops and Den-loops were also found in the circular and catenated ctDNA oligomers from pea and corn plants. Mapping the positions of the D-loops relative to the positions of the Den-loops showed that the structure of the D-loop-containing region in the pea and corn ctDNAs has been conserved to a greater extent than the structure of the rest of the two ctDNA molecules.

The presence of covalently linked ribonucleotides in the closed circular deoxyribonucleic acid from higher plants.

Single-stranded scissions are induced in the covalently closed circular chloroplast (ct-) DNAs from peas, spinach, and lettuce plants by treatment with alkali or by incubation with a mixture of ribonucleases A and T1. These scissions are due to the presence of covalently linked ribonucleotides in these closed circular DNAs. By comparing the scission rates of these ctDNAs to the scission rate of RNA, it has been estimated that pea and spinach ctDNAs contain a maximum of 18 +/- 2 ribonucleotides/molecule, while lettuce ctDNA contains a maximum of 12 +/- 2 ribonucleotides/molecule. Further studies with pea ctDNA by electron microscopic methods have shown that pea ctDNA contains 19 alkali-labile sites at specific locations. A map of the relative positions of the alkali-labile sites has been constructed. These alkali-labile sites are presumably due to the insertion of individual ribonucleotides.

The molecular size and conformation of the chloroplast DNA from higher plants

Covalently closed circular chloroplast DNA (ctDNA) molecules have been isolated from pea, bean, spinach, lettuce, corn and oat plants by ethidium bromide/cesium chloride density-gradient centrifugation. As much as 30–40% of the total ctDNA could be isolated as closed circular DNA molecules and up to 80% of the total ctDNA was found in the form of circular molecules. The size of pea, spinach, lettuce, corn and oat ctDNA relative to an internal standard (ΦX174 replicative form II monomer DNA) was determined by electron microscopy. The ctDNAs showed significant differences in their sizes, and their molecular weights ranged from 85.4 · 10 6 for corn ctDNA to 96.7 · 10 6 for lettuce ctDNA. Each of these ctDNAs contained 3–4% of the circular molecules as circular dimers and 1–2% of the circular molecules as catenated dimers. The molecular complexity of these ctDNAs was studied by renaturation kinetics using T4 DNA as a standard. The molecular weights of the unique sequence of the ctDNAs ranged from 83.7 · 10 6 for oat ctDNA to 93.1 · 10 6 for lettuce ctDNA, which are in excellent agreement with the sizes of the circular ctDNA molecules. No repeating sequences were detected in any of the ctDNAs. The ctDNAs from pea, lettuce, corn, and spinach were studied by thermal denaturation using T4 DNA as a standard. All of the ctDNAs melted more broadly than T4 DNA and they all had a distinctly different T m . For example, the T m of pea ctDNA was 1°C below the T m of T4 DNA and the T m of corn was 1.9°C higher than the T m of T4 DNA.

Denaturation mapping studies on the circular chloroplast deoxyribonucleic acid from pea leaves.

The structure of circular pea chloroplast DNA (ctDNA) has been analyzed by denaturation mapping. All of the pea ctDNA molecules that were examined had identical gross base sequences. Denaturation maps were constructed at denaturation levels of 2.5%, 22%, and 44%. These denaturation maps showed that the circular pea ctDNA contained six small AT-rich regions on one-half of the DNA molecule, and two small GC-rich regions on the other half of the DNA molecule. The structure of pea ctDNA circular dimers was also examined. The results showed that the pea ctDNA circular dimers consisted of two monomer length units integrated in tandem repeat.

Size, conformation and purity of chloroplast DNA of some higher plants

1. Chloroplast DNA of Antirrhinum majus, Oenothera hookeri, Beta vulgaris and Spinacia oleracea band at the same buoyant density of 1.697 g · cm −3 in neutral CsCl equilibrium gradients. The corresponding nuclear DNAs band at 1.691, 1.703, 1.695 and 1.695 g · cm −3, respectively. The purity of chloroplast and nuclear DNA can be assessed objectively only in the cases of Antirrhinum and Oenothera. 2. Electron microscopic analysis of chloroplast DNA, purified in CsCl or CsCl ethidium bromide gradients, revealed up to 80% circular molecules. Of these about 15% were of supertwisted conformation. Best yields of circular molecules were recovered when populations of unbroken chloroplasts were subjected to DNAase and phosphodiesterase treatment, and when the DNA was purified from viscous lysates by centrifugation into a CsCl cushion. Treatment of plastids with DNAase alone did not guarantee complete degradation of nuclear DNA. 3. The average contour length of the open circular chloroplast DNA molecules was basically similar for all four plants. They were 45.9 ± 2.1 μm for Antirrhinum, 45.7 ± 1.9 μm for Spinacia, 44.9 ± 1.7 μm for Beta and 45.2 μm for Oenothera. This is comparable to the size derived for the coding capacity of chloroplast DNA from reassociation experiments. As much as 15% of the total population of circles in chloroplast DNA of Spinacia were circular dimers.

Isolation and molecular weight of circular chloroplast DNA from Euglena gracilis

1. Chloroplast DNA from Euglena gracilis has been examined by electron microscopy and by sedimentation analysis in order to establish its molecular size and in order to determine the feasibility of physical isolation of whole molecules. 2. Using bacteriophage λ DNA as an internal standard, a molecular weight of 9.2 · 10 7 is obtained for chloroplast DNA by comparison of contour lengths of circular λ and circular chloroplast DNA molecules found on identical grids in the electron microscope. Chloroplast DNA molecules were also found in a tightly twisted configuration; this is the first demonstration of supercoiled DNA structures in chloroplast preparations. 3. Sucrose gradient sedimentation analysis of chloroplast DNA with molecular wholes and halves of bacteriophage T7 DNA gives a molecular weight for chloroplast DNA identical to that obtained by electron microscopy. Furthermore, molecular whole and half molecules of chloroplast DNA can be effectively isolated by sucrose gradient sedimentation.