Benzyladenine-induced increase in DNA content per chloroplast in intact bean leaves

Benzyladenine (BA) treatment was found to induce chloroplast DNA (ctDNA) synthesis after it had stopped in primary leaves of light-grown intact bean plants (Phaseolus vulgaris L.). The leaves were treated with BA from 7 days after sowing. Chloroplasts were isolated and the ctDNA content per chloroplast was determined. Chloroplast division occurred until 13 days after sowing in untreated leaves. BA stimulated the division keeping the level of ctDNA content per chloroplast the same as that in the untreated controls. After the division period, the ctDNA content per chloroplast increased in BA-treated leaves, but not in controls. Consequently, ctDNA per leaf (or per cell) increased immediately after the beginning of BA treatment, but remained constant in the control leaves.     

Co-immunolocalization of topoisomerase II and chloroplast DNA in developing,dividing and mature wheat chloroplasts

This paper describes the first localization of immunofluorescence of topoisomerase II in developing chloroplasts. In order to investigate the relationship between topoisomerase II and chloroplast DNA (ctDNA) replication during chloroplast development the 7-day-old wheat leaf was used. Topoisomerase II was immunolabelled and fluorescein tagged and the ctDNA simultaneously stained with 4,6-diamidino-2-phenylindole (DAPI) in the same sections. Topoisomerase II was detected at every stage of chloroplast development and maximal levels of topoisomerase II were found in chloroplasts at the time of ctDNA replication. Topoisomerase II was localized around the plastid periphery, exactly mirroring the position of the ctDNA. After chloroplast division both topoisomerase II and ctDNA are seen to be restricted to small discrete areas within the plastid, but at different sites. These findings strongly suggest a role for topoisomerase II in ctDNA decatenation prior to chloroplast division.     

Copy numbers of chloroplast and nuclear genomes are proportional in mature mesophyll cells of Triticum and Aegilops species

The possibility of estimating the proportion of chloroplast DNA (ctDNA) and nuclear DNA (nDNA) in nucleic-acid extracts by selective digestion with the methylation-sensitive restriction enzyme PstI, was tested using leaf extracts from Spinacia oleracea and Triticum aestivum. Values of ctDNA as percentage nDNA were estimated to be 14.58%±0.56 (SE) in S. oleracea leaves and 4.97%±0.36 (SE) in T. aestivum leaves. These estimates agree well with those already reported for the same type of leaf material. Selective digestion and quantitative dot-blot hybridisation were used to determine ctDNA as percentage nDNA in expanded leaf tissue from species of Triticum and Aegilops representing three levels of nuclear ploidy and six types of cytoplasm. No significant differences in leaf ctDNA content were detected: in the diploids the leaf ctDNA percentage ranged between 3.8% and 5.1%, and in the polyploids between 3.5% and 4.9%. Consequently, nuclear ploidy and nDNA amount were proportional to ctDNA amount (r₍₁₉₎=0.935, P>0.01) and hence to ctDNA copy number in the mature mesophyll cells of these species. There was a slight increase in ctDNA copy numbers per chloroplast at higher ploidy levels. The balance between numbers of nuclear and chloroplast genomes is discussed in relation to polyploidisation and to the nuclear control of ctDNA replication.Abbreviations ctDNA chloroplast DNA - nDNA nuclear DNA - RuBPCase ribulose-1,5-bisphosphate carboxylase - DAPI 4,6-diamidine-2-phenylindole     

Intraspecific variation of chloroplast DNA in Dioscorea bulbifera L.

Summary Restriction fragment length polymorphism (RFLP) analysis of chloroplast (ct) DNAs from 15 accessions of Dioscorea bulbifera collected from Africa and Asia was carried out using the Southern hybridization technique. Eight cloned ctDNA fragments of D. bulbifera and D. opposita, which cover 80% of the total chloroplast genome, were used as the probes to detect variation in ctDNA digested with nine restriction endonucleases. Ten variable sites, located in the large and small single-copy regions, were disclosed among the 15 accessions, of which six showed base substitution and four carried length mutation. Positions of the latter mutations were determined on the physical map of ctDNA. Based on these results, chloroplast genomes of the 15 accessions could be classified into nine types. Their phylogenetic relationships are assumed to be as follows: (1) African and Asian chloroplast genomes diverged from each other at the earliest point in time; (2) E-type chloroplast genome, occurring in the south-east edge of the Asian continent, appears to be the most ancient among all the Asian chloroplast genomes; and (3) four chloroplast genomes, found in Asian insular regions, are probably derived independently from the E-type genome. The discrepancy between the taxonomic relationship and the proposed chloroplast genome phylogeny of the present materials is noted.     

PSIIP680 ChlaAP基因在水稻叶绿体基因组中的定位

本实验总结出一套水稻叶绿体DNA的提取方法,并获得清晰的叶绿体DNA限制性内切酶图谱。Southern杂交结果表明,菠菜PSIIP680ChlaAP基因探针与水稻叶绿体DNA的Pst-1,Pst-14,Pvu-2和Sal-1片段的部分顺序有较高的同源性。根据Hirai和赵衍的水稻叶绿体基因组物理图,可以确定该基因位于紧靠RuBPCaseLS基因,距反向重复区约26kb处。高等植物叶绿体基因组中这种基因排列方式还未见报道。     

Changes in mitochondrial DNA levels during development of pea (Pisum sativum L.)

The percentage of mitochondrial DNA (mtDNA) present in total DNA isolated from pea tissues was determined using labeled mtDNA in reassociation kinetics reactions. Embryos contained the highest level of mtDNA, equal to 1.5% of total DNA. This value decreased in light- and dark-grown shoots and leaves, and roots. The lowest value found was in dark-grown shoots; their total DNA contained only 0.3% mtDNA. This may be a reflection of increased nuclear ploidy levels without concomitant mtDNA synthesis. It was possible to compare the mtDNA values directly with previous estimates of the amount of chloroplast DNA (ctDNA) per cell because the same preparations of total DNA were used for both analyses. The embryo contained 1.5% of both mtDNA and ctDNA; this equals 410 copies of mtDNA and 1200 copies of ctDNA per diploid cell. Whereas mtDNA levels decreased to 260 copies in leaf cells of pea, the number of copies of ctDNA increased to 10300. In addition, the levels of ctDNA in first leaves of dark-grown and light-transferred pea were determined, and it was found that leaves of plants maintained in the dark had the same percentage of ctDNA as those transferred to the light.Abbreviations ctDNA chloroplast DNA - mtDNA mitochondrial DNA     

水稻叶绿体DNA的提取和纯化

以籼稻品种珍讪97B为材料,采用溶液捣碎和不连续蔗糖梯度离心的方法提取了籼稻的叶绿体DNA,DNA经限制性内切酶酶解和琼脂糖胶电泳可以得到清晰的条带,来自蚕豆的核酮糖—1,5—二磷酸羧化氧合酶大亚基基因探针和23SrRNA基因探针可以与酶切条带杂交,由此确定了含这二种基因的BamHI酶切片段。     

Distribution of transfer RNA genes in thePisum sativum chloroplast DNA

Purified chloroplast tRNAs were isolated fromPisum sativum leaves and radioactively labeled at their 3′ end using tRNA nucleotidyl transferase and α³²P-labeled CTP. Pea ctDNA was fragmented using a number of restriction endonucleases and hybridized with thein vitro labeled chloroplast tRNAs by DNA transfer method. Genes for tRNAs have been found to be dispersed throughout the chloroplast genome. A closer analysis of the several hybrid regions using recombinant DNA plasmids have shown that tRNA genes are localized in the chloroplast genome in both single and multiple arrangements. Two dimensional gel electrophoresis of total ct tRNA have identified 36 spots. All of them have been found to hybridize withPisum sativum ctDNA. Using recombinant clones, 30 of the tRNA spots have been mapped inPisum sativum ctDNA.     

Effect of Benzyladenine on Diurnal Changes in DNA Content per Chloroplast and Chloroplast Replication in Intact Bean Leaves

The effect of benzyladenine (BA) on the diurnal changes in DNAand Chl contents per chloroplast and chloroplast replicationin primary leaves of bean plants (Phaseolus vulgaris L.) grownunder a 16 h light/8 h dark cycle was studied. Experiments weremade on primary leaves in the early expansion phase, where celldivision had been completed but chloroplasts were replicating.In untreated controls, chloroplast number, Chl content and freshweight per leaf showed daily periodic changes. Chl content perchloroplast increased in the light period every day, and freshweight per leaf increased most rapidly in the early dark period.Chloroplast number per leaf increased rapidly in the early darkperiod on day 9, though the increase began a little earlierand was less sharp on days 8 and 10. During these periods, DNAcontent per chloroplast was decreasing due to chloroplast divisionas chloroplast DNA (ctDNA) per leaf remained unchanged throughoutthe experimental period. BA induced increases in Chi contentper chloroplast, ctDNA content and fresh weight per leaf within6 h of its application, regardless of whether it was appliedat or 10 h after the beginning of the light period. Applicationof BA at 10 h in the light period shifted the start of chloroplastreplication by 6 h compared to that in untreated controls. However,when BA was applied at the beginning of illumination, the startof chloroplast replication showed the same relative change intime as above. 5-Fluorodeoxyuridine (5-FdU) promptly preventedBA-induced increase in Chl content and chloroplast number perleaf as well as ctDNA content per leaf.     

Plastid DNA diversity in natural populations of Beta maritima showing additional variation in sexual phenotype and mitochondrial DNA

Summary Plants of two natural populations of Beta maritima, characterized by high percentages of male-sterile plants, have been investigated for organelle DNA polymorphism. We confirm the two classes of mitochondrial DNA variation previously described: (i) mitochondrial DNA (mtDNA) type N is associated with male fertility, whereas mtDNA type S can cause cytoplasmic male sterility (CMS); (ii) the 10.4-kb linear plasmid is observed in both types of mitochondria and is not correlated with the cytoplasmic male sterility occurring in this plant material. A third polymorphism is now described for chloroplast DNA (ctDNA). This polymorphism occurs within single populations of Beta maritima. Three different ctDNA types have been identified by HindIII restriction analysis. Among the plants studied, ctDNA type 1 is associated with N mitochondria and type 2 with S mitochondria. Chloroplast DNA type 3 has been found both in a fertile N plant and in a sterile S plant. This finding suggests that the chloroplast DNA polymorphism reported is not involved in the expression of male sterility. A comparison with Beta vulgaris indicates that ctDNA type 3 of Beta maritima corresponds to the ctDNA of fertile sugar beet maintainer lines. The three types of Beta maritima ctDNA described in this study differ from the ctDNA of male-sterile sugar beet.     

龙葵叶绿体ATP合酶α-亚单位基因的克隆

本研究以菠菜叶绿体DNA 2.45kb的SalI片段(含有ATP合酶α-亚单位基因)为探针,从龙英叶绿体DNA BamHI片段文库中筛选出含龙葵叶绿体atpA基因的克隆。通过Southrcn吸印与探针杂交,证明了重组质粒pSB 132的插入片段含有atpA基因。同时将atpA基因定位在龙葵叶绿体DNA SalI、BglI、XhoI和BamHI 4种酶切图谱的限制性片段上。     

A procedure for the extraction of chloroplast DNA from broad-leaved tree species

A method for the extraction of ctDNA from isolated chloroplast was developed. This method is simple and adapted particularly to broad-leaved trees, including sclerophyllous species with high phenolic and polysaccharide contents. This method includes two major steps: first, chloroplasts are isolated in non-aqueous solutions to avoid oxidation and phenolic problems; second, ctDNA is extracted from the chloroplasts using aqueous solutions and specific methods to provide highly purified ctDNA.     

Beta chloroplast genomes: analysis of Fraction I protein and chloroplast DNA variation

Summary The interrelationships of Beta chloroplast genomes have been investigated on the basis of the analysis of Fraction I protein and chloroplast (ct) DNA. Three groups of the chloroplast genomes could be demonstrated by the difference in isoelectric points of the large subunit of Fraction I protein. Restriction enzyme analysis revealed inter- and intra-specific variations among the ctDNAs, which enabled us to detect seven distinct ctDNA types. In Vulgares and Corollinae species, the observed differences were physically mapped taking advantage of the restriction fragment map available for sugar beet (B. vulgaris) ctDNA. The DNA variations were found to result either from gains or losses of restriction sites or from small deletions/ insertions, and most of them were located in the large single-copy region of the genome. Moreover, the ctDNAs from Patellares species are more diverged from those of other Beta taxa. Our results also indicate that there is a close correlation between the chloroplast genome diversity and the accepted taxonomic classification of the species included in this survey.     

Biparental inheritance of chloroplast DNA and the existence of heteroplasmic cells in alfalfa

Summary Mapping of chloroplast DNA (ctDNA) restriction fragment patterns from a chlorophyll deficient mutant and two phenotypically normal alfalfa genotypes (Medicago sativa L.) has demonstrated the existence of a distinct ctDNA genotype from each source. These unique restriction fragment patterns were utilized to identify maternal or paternal origin of ctDNA in hybrid plants from crosses involving the normal alfalfa genotypes as females and the yellow-green chlorophyll deficient sectors as males. Progeny from these crosses expressing the yellow-green sectored phenotypes contained paternal ctDNA in the chlorophyll deficient sectors and maternal ctDNA in the normal sectors, confirming biparental plastid inheritance. The existence of mixed cells containing both mutant and normal plastids at various stages of sorting-out was observed by transmission electron microscopy of mesophyll cells in mosaic tissue from hybrid plants. This observation verified the biparental transmission of plastids in alfalfa.     

Divergence of tRNA genes in chloroplast DNA of higher plants

The saturation hybridization between spinach chloroplast (ct) DNA and spinach ¹²⁵I-labelled chloroplast tRNA has shown that about 1.1% of the spinach ctDNA codes for tRNAs. The observed hybridization is a result of specific base-pairing as shown by competition hybridization experiments and thermal stability of the ctDNA-tRNA hybrids. The amount of hybridization shows that spinach ctDNA contains about 40 tRNA genes. Similar hybridization studies have shown that corn ctDNA contains about 28 tRNA genes. The cross-hybridizations between ctDNA and tRNAs of corn, spinach and pea have shown that tRNAs in chloroplasts of higher plants have undergone significant divergence. The pea and spinach tRNAs have been found to have 50% of the base sequences in common. The corn tRNAs have been found to have only about 30% of the base sequences in common with pea and spinach. These data have been confirmed by extensive heterologous competition experiments and thermal stability of the heterologous DNA-tRNA hybrids. The experiments have also shown that the base sequences of tRNAs common in all three plants are the same.     

Sequence Homology between Chloroplast DNAs from Several Higher Plants

An estimate has been made of the amount of sequence homology present in the chloroplast DNA (ctDNA) of several higher plants by the technique of DNA-DNA hybridization. Approximately 85% of tomato, 60% of spinach, 45% of kale, and 15% of barley ctDNA sequences were found to hybridize with tobacco ctDNA under conditions in which maximum hybridization in homologous reactions reached 85%. All heteroduplexes contained significant amounts of sequence mismatch as indicated by a 3 to 9 C decrease in melting temperature as compared to homoduplex.     

Cultivated potato chloroplast DNA differs from the wild type by one deletion — evidence and implications

Summary The chloroplast DNA (ctDNA) of Solanum tuberosum ssp. tuberosum (T type) and S. chacoense (W type) yield five different restriction fragment patterns with five different restriction endonucleases. DNA-DNA hybridization tests revealed that these differences were all caused by one physical deletion (about 400 bp in size) in the ctDNA of ssp. tuberosum. This suggests that T type ctDNA of the common potato and of Chilean tuberosum originated from W type ctDNA. The deleted region of the T type ctDNA is probably not concerned with gene-cytoplasmic male sterility.Reference to a specific brand or firm name does not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned     

Isolation, characterization and restriction endonuclease mapping of the Petunia hybrida chloroplast DNA

A procedure is developed for the isolation of intact chloroplast DNA (ctDNA) from Petunia hybrida. The molecular weight, calculated from contour length measurements, is 96.0 +/- 4.5 x 10(6) daltons. This value is in good agreement with the value of 101.2 x 10(6) daltons that was estimated from the electrophoretic mobilities of restriction endonuclease fragments of ctDNA. Analysis of petunia ctDNA in neutral CsCl gradients revealed the presence of only one type of DNA at a buoyant density of 1.6987 +/- 0.0005 gcm-3. This corresponds with a GC-content of 39.3 +/- 0.5%. A physical map of petunia ctDNA was constructed by using the restriction endonucleases Sal I, Bgl I, Hpa I and Kpn I. The map indicates that petunia ctDNA contains two copies of a sequence in an inverted orientation. The inverted repeat regions have a minimum length of 10 x 10(6) daltons. Hybridization data indicate that part of the inverted repeat regions contain the genes for chloroplast ribosomal RNAs.     

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.