RESUMO
cDNA corresponding to OsRad51 protein was isolated from cDNA library of rice flowers (Oryza sativa, Indica cultivar group) and cloned in to pET28a expression vector. The protein was over expressed in E. coli BL21 (DE3) and purified. Purified OsRad51 could bind single and double stranded DNA, however it showed higher affinity for single stranded DNA. Transmission Electron Microscopy (TEM) studies of OsRad51-DNA complexes showed that this protein formed ring like structures and bound DNA forming filaments. OsRad51 protein promoted renaturation of complementary single strands in to duplex DNA molecules and also showed ATPase activity, which was stimulated by single strand DNA. Fluorescence resonance energy transfer (FRET) assays revealed that OsRad51 promoted homology dependent renaturation as well as strand exchange reactions. Renaturation activity was ATP dependent; however strand exchange activity was ATP independent. This is the first report on in vitro characterization of Rad51 protein from crop plants.
Assuntos
Oryza/enzimologia , Oryza/genética , Rad51 Recombinase/metabolismo , Recombinação Genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Western Blotting , Cromatografia em Camada Fina , Clonagem Molecular , DNA de Plantas/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Ensaio de Desvio de Mobilidade Eletroforética , Células Eucarióticas/enzimologia , Transferência Ressonante de Energia de Fluorescência , Dados de Sequência Molecular , Ligação Proteica , Renaturação Proteica , Rad51 Recombinase/química , Rad51 Recombinase/isolamento & purificação , Rad51 Recombinase/ultraestrutura , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Using confocal microscopy the organization of tubulin cytoskeleton including endoplasmic and cortical microtubules (CMTs) has been studied in epidermal and cortical cells of the different growth zones of main root of Brassica rapa L. 6-days-old seedlings in control conditions and under clinorotation. It was shown that changes in CMTs orientation occured only in the distal elongation zone (DEZ). In the control, CMT arrays oriented transversely to the root long axis. Under clinorotation appearance of the shorter randomly organized CMTs was observed. Simultaneously, a significant decrease in the cell length in the central elongation zone (CEZ) under clinorotation was detected. It is suggested that the decline of anisotropic growth typical for CEZ cells is connected with CMTs disorientation under clinorotation.
Assuntos
Brassica rapa/ultraestrutura , Microtúbulos/ultraestrutura , Epiderme Vegetal/ultraestrutura , Raízes de Plantas/ultraestrutura , Brotos de Planta/ultraestrutura , Brassica rapa/crescimento & desenvolvimento , DNA de Plantas/ultraestrutura , Gravitação , Gravitropismo , Microscopia Confocal , Epiderme Vegetal/genética , Epiderme Vegetal/crescimento & desenvolvimento , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Rotação , Simulação de Ausência de PesoRESUMO
Mitochondrial nucleoids isolated from mung bean seedlings exhibited a chromatin-like structure associated with a membrane component. A similar structure, which underwent discrete changes during cotyledon development, was identified in situ. Isolated nucleoids consisted of essentially the same phospholipids, including cardiolipin, as whole mitochondria and proteins of inner- and outer-mitochondrial-membrane origin. Actin was consistently found with mitochondrial nucleoids prepared with different detergent concentrations. Formaldehyde cross-linking of cytochalasin B- and proteinase K-treated mitochondria further revealed that actin was associated with DNA in nucleoids. Mitochondrial nucleoids were self-sufficient in directing DNA synthesis in vitro in a pattern mimicking mtDNA synthesis in isolated mitochondria. In pulse-field gel electrophoresis, newly synthesized mtDNA separated into two major components, well-bound and fast-moving forms. Nucleoids DNA synthesis was resistant to aphidicolin but sensitive to N-ethylmaleimide, which indicates that a gamma-type DNA polymerase was responsible for this activity. Mitochondrial nucleoids were capable of self-directed RNA transcription in a non-random fashion in vitro. Consistent with and complementary to results from fungi and human cells done mostly in situ, our present work helps to establish the important paradigm that mitochondrial nucleoids in eukaryotes are more than mere mtDNA compaction and segregation entities but are centers of mtDNA maintenance and expression.
Assuntos
DNA Mitocondrial/biossíntese , DNA Mitocondrial/ultraestrutura , Fabaceae/genética , Mitocôndrias/genética , Cromatina/ultraestrutura , DNA Mitocondrial/química , DNA de Plantas/biossíntese , DNA de Plantas/química , DNA de Plantas/ultraestrutura , Fabaceae/ultraestrutura , Membranas Intracelulares/química , Mitocôndrias/química , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/análise , Transcrição GênicaRESUMO
The molecular events of recombination are thought to be catalyzed by proteins present in recombination nodules (RNs). Therefore, studying RN structure and function should give insights into the processes by which meiotic recombination is regulated in eukaryotes. Two types of RNs have been identified so far, early (ENs) and late (LNs). ENs appear at leptotene and persist into early pachytene while LNs appear in pachytene and remain into early diplotene. ENs and LNs can be distinguished not only on their time of appearance, but also by such characteristics as shape and size, relative numbers, and association with unsynapsed and/or synapsed chromosomal segments. The function(s) of ENs is not clear, but they may have a role in searching for DNA homology, synapsis, gene conversion and/or crossing over. LNs are well correlated with crossing over. Here, the patterns of ENs and LNs during prophase I in plants are reviewed.
Assuntos
Plantas/genética , Recombinação Genética , DNA de Plantas/genética , DNA de Plantas/ultraestrutura , Processamento de Imagem Assistida por Computador , Mutação , Proteínas de Plantas/genéticaRESUMO
We propose that chromosome function is governed by internal mechanical forces generated by programmed tendencies for expansion of the DNA/chromatin fiber against constraining features.
Assuntos
Cromossomos/química , Cromossomos/metabolismo , Animais , Cromatina/química , Cromatina/metabolismo , Cromatina/ultraestrutura , Cromossomos/ultraestrutura , DNA de Plantas/química , DNA de Plantas/metabolismo , DNA de Plantas/ultraestrutura , Humanos , Meiose , Mitose , Modelos Biológicos , Conformação de Ácido Nucleico , Estresse Mecânico , Fatores de TempoRESUMO
The conventional protocol for isolation of cell wall free nuclei for release of DNA fibers for plants involves mechanical removal of the cell wall and separation of debris by sieve filtration. The mechanical grinding pressure applied during the process leaves only the more tolerant G(1) nuclei intact, and all other states of active nuclei that may be present in the target tissues (e.g., leaf) are simply crushed/disrupted during the isolation process. Here we describe an alternative enzymatic protocol for isolation of nuclei from root tip tissue. Cell wall free nuclei at a given stage of cell cycle, free of any cell debris, could be realized in suspension that are fit for preparation of extended fibers suitable for fiber FISH applications. The protocol utilizes selective harvest of active nuclei from root tip tissue in liquid suspension under the influence of cell wall-degrading enzymes, and provides opportunities to target cell cycle-specific nuclei from interphase through division phase for the release of extended DNA fibers. Availability of cell cycle-specific fibers may have added value in transcriptional analysis, DNA:RNA hybridization, visualization of DNA replication and replication forks, and improved FISH efficiency.
Assuntos
Cromatina/isolamento & purificação , DNA de Plantas/isolamento & purificação , Hibridização in Situ Fluorescente/métodos , Ciclo Celular/fisiologia , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Parede Celular/química , Cromatina/genética , Cromatina/metabolismo , DNA de Plantas/genética , DNA de Plantas/metabolismo , DNA de Plantas/ultraestrutura , Magnoliopsida/química , Raízes de Plantas/química , Raízes de Plantas/crescimento & desenvolvimento , RNA Ribossômico 5S/química , RNA Ribossômico 5S/genética , Sensibilidade e EspecificidadeRESUMO
Fluorescence in situ hybridization (FISH) is widely used in molecular biological study. However, high-resolution analysis of fluorescent signals is theoretically limited by the 300-nm resolution optical limit of light microscopy. As an alternative to detection by light microscopy, we used Scanning Near-field Optical/Atomic Force Microscopy (SNOM/AFM), which can simultaneously obtain topographic and fluorescent images with nanometer-scale resolution. In this study, we demonstrated high-resolution SNOM/AFM imaging of barley chromosome (Hordeum vulgare, cv. Minorimugi) FISH signals using telomeric DNA probes. Besides detecting the granular structures on chromosomes in a topographic image, we clearly detected fluorescent signals in telomeric regions with low-magnification imaging. The high-resolution analysis suggested that one of the telomeric signals could be observed by expanded imaging as two fluorescent regions separated by approximately 250 nm. This result indicated that the fluorescent signals beyond the optical limit were detected with higher resolution scanning by SNOM/AFM.
Assuntos
Cromossomos/ultraestrutura , Hibridização in Situ Fluorescente/métodos , Microscopia de Força Atômica/métodos , Cromossomos/genética , Sondas de DNA , DNA de Plantas/genética , DNA de Plantas/ultraestrutura , Regulação da Expressão Gênica de Plantas/genética , Hordeum/genética , Hordeum/ultraestrutura , Hibridização in Situ Fluorescente/instrumentação , Microscopia de Força Atômica/instrumentação , Sensibilidade e Especificidade , Telômero/genética , Telômero/ultraestruturaRESUMO
Nano-scale structures of the YOYO-1-stained barley chromosomes and lambda-phage DNA were investigated by scanning near-field optical/atomic force microscopy (SNOM/AFM). This technique enabled precise analysis of fluorescence structural images in relation to the morphology of the biomaterials. The results suggested that the fluorescence intensity does not always correspond to topographic height of the chromosomes, but roughly reflects the local amount and/or density of DNA. Various sizes of the bright fluorescence spots were clearly observed in fluorescence banding-treated chromosomes. Furthermore, fluorescence-stained lambda-phage DNA analysis by SNOM/AFM demonstrated the possibility of nanometer-scale imaging for a novel technique termed nano-fluorescence in situ hybridization (nano-FISH). Thus, SNOM/AFM is a powerful tool for analyzing the structure and the function of biomaterials with higher resolution than conventional optical microscopes.
Assuntos
Bacteriófago lambda/genética , Cromossomos de Plantas/genética , DNA de Plantas/ultraestrutura , DNA Viral/ultraestrutura , Hordeum/genética , Microscopia de Força Atômica/métodos , Benzoxazóis/metabolismo , DNA de Plantas/genética , DNA Viral/genética , Corantes Fluorescentes/metabolismo , Nanotecnologia , Compostos de Quinolínio/metabolismoRESUMO
We have identified a putative homologue of the KU70 gene (AtKU70) from Arabidopsis thaliana. In order to study its function in plants we have isolated an A.thaliana line that contains a T-DNA inserted into AtKU70. Plants homozygous for this insertion appear normal and are fertile. In other organisms the KU70 gene has been shown to play a role in the repair of DNA damage induced by ionising radiation (IR) and by radiomimetic chemicals such as methylmethane sulfonate (MMS). We show that AtKU70(-/-) plants are hypersensitive to IR and MMS, and thus the AtKU70 gene plays a similar role in DNA repair in plants as in other organisms. The KU70 gene also plays a role in maintaining telomere length. Yeast and mammalian cells deficient for Ku70 have shortened telomeres. When we studied the telomeres in the AtKU70(-/-) plants we found unexpectedly that they were significantly longer (>30 kb) than was found in wild-type plants (2-4 kb). We propose several hypotheses to explain this telomere lengthening in the AtKU70(-/-) plants.
Assuntos
Antígenos Nucleares , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Arabidopsis/ultraestrutura , Dano ao DNA , DNA Helicases , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas de Saccharomyces cerevisiae , Telômero/ultraestrutura , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/efeitos da radiação , DNA de Plantas/ultraestrutura , Genes de Plantas , Autoantígeno Ku , Metanossulfonato de Metila/farmacologia , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Tolerância a Radiação , Alinhamento de SequênciaRESUMO
Apoptosis was observed in the initial leaf of 5-8-day-old etiolated wheat seedlings. A condensation of cytoplasm in apoptotic cells, formation of myelin-like structures, specific fragmentation of cytoplasm, appearance in vacuoles of specific vesicles containing subcellular organelles, condensation and margination of chromatin in the nucleus, and internucleosomal fragmentation of nuclear DNA are ultrastructural features of apoptosis in the initial wheat leaf. Single-membrane vesicles detected in vacuoles of the leaf cells resemble in appearance the vacuolar vesicles in the coleoptile apoptotic cells described earlier (Bakeeva, L. E., et al. (1999) FEBS Lett., 457, 122-125); they contain preferentially plastids but not mitochondria as was observed in coleoptile. The vacuolar vesicles are specific for the apoptotic plant cells. Thus, apoptosis in various tissues is an obligatory element of plant (wheat) growth and development even in the early stages of ontogenesis. Contrary to strong geroprotecting action in coleoptile, the known antioxidant BHT (ionol, 2.27 x 10(-4) M) does not prevent in the leaf cells the apoptotic internucleosomal DNA fragmentation and appearance of specific vacuolar vesicles containing subcellular organelles. Therefore, the antioxidant action on apoptosis in plants is tissue specific. Peroxides (H2O2, cumene hydroperoxide) stimulated apoptosis (internucleosomal DNA fragmentation) in coleoptile and induced it in an initial leaf when apoptosis in a control seedling leaf was not yet detected. Thus, apoptosis that is programmed in plant ontogenesis and controlled by reactive oxygen species (ROS) can be modulated by anti- and prooxidants.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Derivados de Benzeno/farmacologia , Hidroxitolueno Butilado/farmacologia , Peróxido de Hidrogênio/farmacologia , Triticum/efeitos dos fármacos , Divisão Celular , Núcleo Celular , DNA de Plantas/ultraestrutura , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/ultraestrutura , Plantas/efeitos dos fármacos , Plantas/ultraestrutura , Triticum/ultraestruturaRESUMO
Contemporary data on ultrastructural reconstruction and changes in functional nucleolus activity in plant cells affected by physical environmental factors are presented. The main attention is paid to modifications of ribosomal gene expression under these conditions and induced changes in r-chromatin structure and ribosomal DNA localization. Recent data on fine nucleolus structure and molecular aspects of its organization as well as influence of microgravitation and clinostating on structural and functional organization of plant nucleoli are reported.
Assuntos
Nucléolo Celular/metabolismo , Meio Ambiente , Plantas/ultraestrutura , Adaptação Biológica , Nucléolo Celular/genética , Nucléolo Celular/ultraestrutura , Cromatina/química , Cromatina/ultraestrutura , DNA de Plantas/ultraestrutura , DNA Ribossômico/ultraestrutura , Expressão Gênica , Gravitação , Microscopia Eletrônica , Fenômenos Fisiológicos Vegetais , Ribossomos/metabolismo , Ribossomos/ultraestruturaRESUMO
Aegilops caudata L. is a diploid wild relative of wheat distributed over the north-eastern Mediterranean from Greece to northern Iraq. To elucidate the geographical differentiation pattern, 35 accessions derived from the entire distribution area were crossed with four Tester strains. Pollen fertility in the F1 hybrids varied from 0 to 96.3% among cross combinations, closely correlating with the geographical regions where the parental accessions were collected. Based on the intraspecific hybrid sterility, the present distribution area of Ae. caudata was divided into two geographical regions effectively isolated by the mountainous region lying between West Anatolia and Central Anatolia. The western region is composed of Greece and West Anatolia, while the eastern region consists of Central Anatolia, South Anatolia, East Anatolia and northern Iraq. The present results and the facts from recent palaeopalynological works suggest that during the maximum glacial period from 18,000 BP to 16,000 BP, Ae. caudata occurred in the two isolated regions, i.e., the region surrounding the Aegean Sea and the western Levant or some sheltered habitats in the East Taurus/Zagros mountains arc, and that it migrated into Central and East Anatolia from the latter regions as the climate became warmer. Furthermore, it is also suggested that the Levant populations now occur in the eastern region of the distribution, while those occurring in the Aegean Sea region during the last glacial period now occupy the western region of the distribution.
Assuntos
Geografia , Triticum/crescimento & desenvolvimento , Cromossomos/ultraestrutura , DNA de Plantas/ultraestrutura , Fertilidade , Pool Gênico , Grécia , Hibridização Genética , Iraque , Metáfase , Filogenia , Pólen/fisiologia , Sementes/fisiologia , Triticum/classificação , Triticum/genética , TurquiaRESUMO
A new family of transposons, FARE, has been identified in Arabidopsis. The structure of these elements is typical of foldback transposons, a distinct subset of mobile DNA elements found in both plants and animals. The ends of FARE elements are long, conserved inverted repeat sequences typically 550 bp in length. These inverted repeats are modular in organization and are predicted to confer extensive secondary structure to the elements. FARE elements are present in high copy number, are heterogeneous in size, and can be divided into two subgroups. FARE1's average 1.1 kb in length and are composed entirely of the long inverted repeats. FARE2's are larger, up to 16.7 kb in length, and contain a large internal region in addition to the inverted repeat ends. The internal region is predicted to encode three proteins, one of which bears homology to a known transposase. FARE1.1 was isolated as an insertion polymorphism between the ecotypes Columbia and Nossen. This, coupled with the presence of 9-bp target-site duplications, strongly suggests that FARE elements have transposed recently. The termini of FARE elements and other foldback transposons are imperfect palindromic sequences, a unique organization that further distinguishes these elements from other mobile DNAs.
Assuntos
Arabidopsis/genética , DNA de Plantas/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Pareamento de Bases , Sequência de Bases , Sequência Consenso , Elementos de DNA Transponíveis/genética , DNA de Plantas/ultraestrutura , Duplicação Gênica , Dados de Sequência Molecular , Família Multigênica , Conformação de Ácido Nucleico , Filogenia , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
We have studied intermediates of the recombination and replication of chromosomal mitochondrial (mt) DNA prepared from suspension cultured cells of Chenopodium album (L.) by electron microscopy during the whole growth cycle. We identified several types of potential recombination and replication intermediates including rosette-like structures, as well as other branched and sigma-like molecules. The absolute and relative amounts of these structures changed dramatically during the growth cycle, indicating high dynamics in the structural organization of the mt genome. The rosette-like molecules had sizes of 2-5 genome units and were found to contain putative replication forks and 'Holliday'-junctions known from recombination intermediates. The high number of rosettes during the first days of culture, and their drastic reduction in the stationary growth stage, were found to be inversely related to changes in the quantity of linear molecules of 40-200 kb. This observation suggests that linear molecules participate in the formation of giant branched rosette-like structures. Most linear molecules were previously found to have at least one single-stranded end, which may allow recombinative invasion of other double-stranded molecules. Thus, recombination events may lead to the formation of more complex molecules and initiate replication similar to phage T4. We propose the coexistence of a recombination-dependent mode of replication with a presumably recombination-independent rolling-circle mode of replication in the mitochondria of C. album.
Assuntos
Bacteriófago T4/genética , Chenopodiaceae/genética , Replicação do DNA , DNA Mitocondrial/ultraestrutura , Mitocôndrias/genética , Recombinação Genética , Bacteriófago T4/ultraestrutura , DNA Circular/ultraestrutura , DNA Mitocondrial/genética , DNA de Plantas/genética , DNA de Plantas/ultraestrutura , Microscopia Eletrônica , Mitocôndrias/ultraestruturaRESUMO
The recovery of maize (Zea mays L.) chromosome addition lines of oat (Avena sativa L.) from oat x maize crosses enables us to analyze the structure and composition of individual maize chromosomes via the isolation and characterization of chromosome-specific cosmid clones. Restriction fragment fingerprinting, sequencing, and in situ hybridization were applied to discover a new family of knob associated tandem repeats, the TR1, which are capable of forming fold-back DNA segments, as well as a new family of centromeric tandem repeats, CentC. Analysis of knob and centromeric DNA segments revealed a complex organization in which blocks of tandemly arranged repeating units are interrupted by insertions of other repeated DNA sequences, mostly represented by individual full size copies of retrotransposable elements. There is an obvious preference for the integration/association of certain retrotransposable elements into knobs or centromere regions as well as for integration of retrotransposable elements into certain sites (hot spots) of the 180-bp repeat. DNA hybridization to a blot panel of eight individual maize chromosome addition lines revealed that CentC, TR1, and 180-bp tandem repeats are found in each of these maize chromosomes, but the copy number of each can vary significantly from about 100 to 25,000. In situ hybridization revealed variation among the maize chromosomes in the size of centromeric tandem repeats as well as in the size and composition of knob regions. It was found that knobs may be composed of either 180-bp or TR1, or both repeats, and in addition to large knobs these repeated elements may form micro clusters which are detectable only with the help of in situ hybridization. The association of the fold-back elements with knobs, knob polymorphism and complex structure suggest that maize knob may be consider as megatransposable elements. The discovery of the interspersion of retrotransposable elements among blocks of tandem repeats in maize and some other organisms suggests that this pattern may be basic to heterochromatin organization for eukaryotes.
Assuntos
Centrômero/ultraestrutura , Cromátides/ultraestrutura , Cromossomos/ultraestrutura , Zea mays/ultraestrutura , Avena/genética , Avena/ultraestrutura , Centrômero/genética , Cromátides/genética , Cromossomos/genética , DNA de Plantas/genética , DNA de Plantas/ultraestrutura , Heterocromatina/genética , Heterocromatina/ultraestrutura , Hibridização Genética , Retroelementos/genética , Sequências de Repetição em Tandem/genética , Zea mays/genéticaRESUMO
Thirty-six accessions, representing the full complement of all the nine annual Cicer L. species, viz C. arietinum, C. reticulatum, C. echinospermum, C. pinnatifidum, C. judaicum, C. bijugum, C. chorassanicum, C. yamashitae and C. cuneatum, were subjected to karyotype analysis for the first time in a single comprehensive study. The detailed karyotype of C. chorassanicum was also investigated for the first time. A 12 h cold water pretreatment and 13 min 60 degrees C 1 N HCl hydrolysis confirmed a somatic chromosome number of 2n = 16 in all the species. Within species interchromosomal size variation was observed to be quite large in C. arietinum, C. reticulatum and C. echinospermum, but not in the remaining six species. Individual chromosome size ranged from 3.77 microns in C. echinospermum to 1.32 microns in C. arietinum while the haploid genome length ranged from 20.65 microns in C. echinospermum to 14.92 microns in C. cuneatum. Ample rearrangement of chromatin among chromosomes within a species was implied to have played a role in Cicer genome evolution. The nine species were classified in two groups based on karyotypic similarity, with the first group comprising the inter-crossable species C. arietinum, C. reticulatum and C. echinospermum, while the remaining species forming the second group. The first group species are also genetically close to each other as deduced by other morphological, biochemical and DNA based studies. Circumstantial evidence has lead to the speculation that perhaps karyotypic similarity and interspecific crossability are positively related to each other.
Assuntos
Cromossomos/ultraestrutura , DNA de Plantas/ultraestrutura , Fabaceae/genética , Plantas Medicinais , Centrômero/ultraestrutura , DNA Satélite/ultraestrutura , Diploide , Heterocromatina/ultraestrutura , Cariotipagem , Especificidade da EspécieRESUMO
The structure of sigma-like mitochondrial DNA molecules prepared from suspension cultured cells of Chenopodium album (L.) was studied by electron microscopy. These molecules were highly variable in size, ranging from about 1 to 104 kb, and had single- and double-stranded regions typical for rolling circle replicating intermediates. Partial denaturation studies confirmed that these structures constitute rolling circles. Close inspection of the circle-tail junctions of the replication fork at high magnification suggests that in circles with a double-stranded tail, both strands of the tail seem to be covalently attached to the circle in about 27% of the molecules. This observation can be explained by a phenomenon called strand switching or strand splippage during rolling circle replication, similar to a mechanism proposed for bacterial replicons or in vitro replicating constructs harboring bacteriophage T4 replication origins.
Assuntos
Chenopodiaceae/genética , Replicação do DNA , DNA Circular/biossíntese , DNA Mitocondrial/biossíntese , DNA de Plantas/biossíntese , DNA de Cadeia Simples/biossíntese , Plasmídeos/metabolismo , Chenopodiaceae/ultraestrutura , DNA Circular/ultraestrutura , DNA Mitocondrial/ultraestrutura , DNA de Plantas/ultraestrutura , DNA de Cadeia Simples/ultraestrutura , Eletroforese em Gel de Campo Pulsado , Microscopia Eletrônica , Plasmídeos/ultraestruturaRESUMO
According to the endosymbiotic theory, mitochondrial genomes evolved from the chromosome of an alpha-proteobacterium-like ancestor and developed during evolution an extraordinary variation in size, structure and replication. We studied in vitro DNA replication of the mitochondrial circular plasmid mp1 (1309 bp) from the higher plant Chenopodium album (L.) as a model system that replicates in a manner reminiscent of bacterial rolling circle plasmids. Several mp1 subclones were tested for their ability to support DNA replication using a newly developed in vitro system. Neutral/neutral two-dimensional gel electrophoresis of the in vitro products revealed typical simple Y patterns of intermediates consistent with a rolling circle type of replication. Replication activity was very high for a BamHI-restricted total plasmid DNA clone, a 464 bp BamHI/KpnI fragment and a 363 bp BamHI/SmaI fragment. Further subcloning of a 148 bp BamHI/EcoRI fragment resulted in the strongest in vitro DNA replication activity, while a 1161 bp-template outside of this region resulted in a substantial loss of activity. Electron microscopic studies of in vitro DNA replication products from the highly active clones also revealed sigma-shaped molecules. These results support our in vivo data for the presence of a predominant replication origin between positions 628 and 776 on the plasmid map. This sequence shares homology with double-stranded rolling circle origin (dso) or transfer origin (oriT) nicking motifs from bacterial plasmids. mp1 is the first described rolling circle plasmid in eukaryotes.
Assuntos
Chenopodiaceae/metabolismo , Replicação do DNA , DNA Mitocondrial/biossíntese , DNA de Plantas/biossíntese , Sequência de Bases , Chenopodiaceae/genética , Conjugação Genética , DNA Bacteriano/biossíntese , DNA Circular/biossíntese , DNA Mitocondrial/genética , DNA Mitocondrial/ultraestrutura , DNA de Plantas/genética , DNA de Plantas/ultraestrutura , Microscopia Eletrônica , Plasmídeos/biossíntese , Plasmídeos/genética , Plasmídeos/ultraestrutura , Origem de Replicação , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
Mitochondrial (mt) DNA of higher plants is unique in its large size and complexity. We report here a hitherto unknown feature, the presence of large quantities of single-stranded (ss) DNA. About 2.0-8.5% of the chromosomal mtDNA from a suspension culture (depending on the growth stage) and 6.5% of the chromosomal mtDNA from whole plants of Chenopodium album were found to be in ss form by dot-blot hybridization after neutral transfer. Similar amounts of ss mtDNA were observed by binding of the single-strand binding (SSB) protein of Escherichia coli under the electron microscope. Significantly less ssDNA was found in plastids of C. album and in E. coli cells. We observed ss regions between 100 and 22,800 bases distributed in the mt genome spaced from 0.5-100 kb apart. After pulsed-field gel electrophoresis (PFGE), the well-bound fraction of mtDNA (found to consist of circular, sigma-shaped and rosette-like molecules), contained the major part of ssDNA as opposed to the migrating linear molecules. Digestion of mtDNA by ss-specific nucleases followed by PFGE mobilized all well-bound DNA and correspondingly increased the quantity of migrating linear DNA molecules. The implications of ssDNA for the structural organization on plant mt genomes are discussed.
Assuntos
DNA Mitocondrial/análise , DNA de Plantas/análise , DNA de Cadeia Simples/análise , Mitocôndrias/química , Plantas/química , DNA Mitocondrial/química , DNA Mitocondrial/ultraestrutura , DNA de Plantas/química , DNA de Plantas/ultraestrutura , DNA de Cadeia Simples/química , DNA de Cadeia Simples/ultraestrutura , Proteínas de Ligação a DNA , Desoxirribonucleases , Eletroforese em Gel de Campo Pulsado , Peso Molecular , Plastídeos/químicaRESUMO
A protein with structure-specific endonuclease activity has been purified to near homogeneity from cauliflower ( Brassica oleracea var. botrytis) inflorescence through five successive column chromatographies. The protein is a single polypeptide with a molecular mass of 40 kDa. Using three different branched DNA structures (flap, pseudo-Y and stem-loop) we found that the enzyme, a cauliflower structure-specific endonuclease, cleaved the single-stranded tail in the 5'-flap and 5'-pseudo-Y structures, whereas it could not incise the 3'-flap and 3'-pseudo-Y structures. The incision points occur around the single strand-duplex junction in these DNA substrates and the enzyme leaves 5'-PO4 and 3'-OH termini on DNA. The protein also endonucleolytically cleaves on the 3'-side of the single-stranded region at the junction of unpaired and duplex DNA in the stem-loop structure. The structure-specific endonuclease activity is stimulated by Mg2+ and by Mn2+, but not by Ca2+. Like mammalian FEN-1, the protein has weak 5'-->3' double-stranded DNA-specific exonuclease activity. These results indicate that the cauliflower protein is a plant structure-specific endonuclease like mammalian FEN-1 or may be the plant alternative.