The organization of mitochondrial atp6 gene region in male fertile and CMS lines of pepper (Capsicum annuum L.)

The mitochondrial atp6 gene in male fertile (N) and CMS (S) pepper has previously been compared and was found to be present in two copies (Kim et al. in J Kor Soc Hort Sci 42:121–127 2001). In the current study, these atp6 copies were amplified by an inverse PCR technique, and the coding region as well as the 5′ and 3′ flanking regions were sequenced. The atp6 copies in CMS pepper were detected as one intact gene and one pseudogene, truncated at the 3′ coding region. When the atp6 genes in pepper were compared to other plant species, pepper, potato, and petunia all possessed a sequence of 12 identical amino acids at the 3′ extended region, which was considered a hallmark of the Solanaceae family. Northern blot analysis showed differences in mRNA band patterns between CMS and restorer lines, indicating that atp6 gene is one of the candidates for CMS in pepper. GenBank accession number: DQ126682 (atp6-1 genomic sequence common to fertile and CMS pepper), DQ126681 (Fertile atp6-2 genomic sequence), DQ126680 (CMS pseudo-atp6-2 genomic sequence)     

Organization of ATPA coding and 3′ flanking sequences associated with cytoplasmic male sterility in Phaseolus vulgaris L.

Summary A region of the mitochondrial genome associated with cytoplasmic male sterility (CMS) in Phaseolus vulgaris was flanked by two different repeated sequences designated x and y. The DNA sequence of the CMS-unique region and a portion of each flanking repeat was determined. Repeat x contained a complete coding copy of the F1 ATPase subunit A (atp A) gene, as well as an open reading frame (orf) predicting a protein of 209 amino acids. The TGA termination codon of the atpA gene and the ATG initiation codon of orf209 were overlapping. These reading frames were oriented with their 3 ends proximal to the CMS-unique region. The CMS-unique region of 3736 nucleotides contained numerous orfs. The longest of these predicted proteins being of 239, 98 and 97 amino acids. The 3 coding and 3 flanking regions of orf98 were derived from an internal region of the higher plant chloroplast tRNA alanine intron. The region of repeat y immediately adjacent to the CMS-unique region contained the 111 carboxy-terminal coding residues of the apocytochrome b (cob) gene. This segment was oriented with its 5 end proximal to the CMS-unique region, but cob gene sequences were not fused to an initiation codon within the unique region.     

A mitochondrial plasmid specifically associated with male sterility and its relation with other mitochondrial plasmids in Vicia faba L.

Summary Small supercoiled DNA molecules of Vicia faba were cloned and characterized by restriction mapping and molecular hybridization. The 1700 by S plasmid has been found to be specifically associated with the male sterile cytoplasms and exhibited large homologies with the other 1700 by F plasmid as shown by molecular cross hybridization. In one line bearing the male sterile 350 cytoplasm, a deleted mitochondrial plasmid of 1540 bp was observed and this plasmid derives from the 1700 bp S plasmid. Our results indicate therefore that three of the four plasmids analysed are closely related between each other.     

Properties and nucleotide sequence of a mitochondrial plasmid from sunflower

Summary The 1.413 circular supercoiled mitochondrial DNA plasmid P 1 from a fertile sunflower line was sequenced, and a series of 160 by tandemly repeated sequences was observed. The P1 plasmid was detected in both fertile and cytoplasmic male-sterile (CMS) lines, but in different quantities. Two other circular plasmids, P2 and P3, each 1.8 kbp in length, were shown to share common sequences with Pl. The mitochondrial plasmid P1 detected homologous sequences in the nuclear DNA of sunflower, but not in chloroplast DNA nor in main band mitochondrial DNA. RNA molecules of about 680 and 550 nucleotides were detected that were complementary to mt plasmid P1.     

Mobilization and evolutionary history of miniature inverted-repeat transposable elements (MITEs) in Beta vulgaris L.

We have identified three families of miniature inverted-repeat transposable elements (VulMITEs) in the genome of sugar beet (Beta vulgaris L.), evidently derived from a member of the Vulmar family of mariner transposons. While VulMITEs I are typical stowaway-like MITEs, VulMITEs II and VulMITEs III are rearranged stowaway elements of increased size. The integration of divergent moderately and highly repetitive sequences into VulMITEs II and, in particular in VulMITEs III, respectively, shows that amplification of repetitive DNA by MITEs contribute to the increase of genome size with possible implications for plant genome evolution. Fluorescent in-situ hybridization (FISH), for the first time visualizing stowaway MITE distribution on plant chromosomes, revealed a dispersed localization of VulMITEs along all B. vulgaris chromosomes. Analysis of the flanking sequences identified a dispersed repeat as target site for the integration of the stowaway element VulMITE I. Recent transposition of VulMITE I, which most likely occurred during the domestication of cultivated beets, was concluded from insertional polymorphisms between different B. vulgaris cultivars and species. ^†Sequence data from this article have been deposited in the EMBL/GenBank Data Library under the accession nos. AM231630-AM231653 and AM259123-AM259125.     

Ultrastructure of giant and small thalamic terminals of cortical origin: a study of the projections from the barrel cortex in mice using Phaseolus vulgaris leuco-agglutinin (PHA-L)

Summary By means of tracing with the lectin Phaseolusvulgaris leucoagglutinin (PHA-L), we examined in the thalamus of the mouse, the axon terminals of fibers originating in the barrel cortex. Vibratome sections of the brain were subjected to PHA-L immunocytochemistry and processed for light and electron microscopy. We observed small (0.5–0.8 m in diameter) varicosities of labeled fibers in the nucleus ventrobasalis (VB) and the nucleus posterior (PO) as well as labeled giant terminals (3–5 m in diameter) in PO. The analysis involved examination of serial sections and computer-aided reconstruction of several terminals. The small varicosities in VB appear to be small axon terminals forming distinct asymmetric synapses with small dendritic profiles. Some labeled terminals are apposed to, but not synaptically related with, the cell bodies of neurons in VB that are retrogradely labeled with PHA-L. The small varicosities seen with the light microscope in PO are terminals forming asymmetric synapses with dendritic shafts. The giant terminals in PO appear as large, vesicle-filled profiles forming part of synaptic glomeruli, i.e. complexes of one corticothalamic terminal engulfing several excrescences of a single dendrite. A giant terminal forms several asymmetric synapses (about 8) with these excrescences, as well as numerous (up to 15) puncta adhaerentia. The glomeruli are enveloped in glial lamellae, and they are often found at the bifurcations of primary dendritic segments. We suggest that the small terminals in VB are in the service of feedback signalling from the barrel cortex to its principal thalamic relay nucleus; the functional importance of this projection may reside in increased spatio-temporal discrimination. We interpret the giant terminals in PO as elements serving feed-forward processing, allowing the barrel cortex to influence, via PO, parts of the motor pathway modulating the animal's ongoing behavior.     

Mitochondrial DNA of Chenopodium album (L): a comparison of leaves and suspension cultures

Summary The mitochondrial genome (mt genome) of Chenopodium album was analysed. It was found to have a size of about 270 kb and a GC content of 46%. The genomes of plant cells and suspension cultures were compared. Restriction fragment pattern analyses and hybridization experiments revealed quantitative and qualitative alterations. However, a comparison of the restriction fragment patterns of two independently established in vitro cultures did not reveal differences as far as the mt chromosomal DNA is concerned. Therefore, alterations in mtDNA induced by in vitro culture seem not to be caused by an entirely undirected process in Chenopodium. A plasmid-like DNA molecule was amplified in only one of the cultures investigated and not in the other or in the plant cells. This molecule has a length of 1,083 by and is referred to as Ca1 mp1.     

FISH to mitotic chromosomes and extended DNA fibres of Beta procumbens in a series of monosomic additions to beet (B. vulgaris)

The physical localization and organization of a Procumbentes-specific repetitive DNA sequence, PB6-4, on the chromosomes of Beta procumbens (2n=18) were studied, using FISH (fluorescence in situ hybridization) to mitotic chromosomes and extended DNA fibres. The chromosomes of B. procumbens were studied in metaphase complements of the species itself, as well as in preparations of a series of eight different B. procumbens-derived monosomic additions to B. vulgaris (2n=18). FISH to chromosome spreads of B. procumbens revealed that PB6-4 hybridizes to all chromosomes, predominantly in the pericentromeric regions, but with differences in size and brightness of the signals. Hybridization of PB6-4 to metaphase complements of B. vulgaris revealed no signals, indicating that cross-hybridization with the genome of this species was negligible. Consequently, hybridization of PB6-4 to metaphase complements of the monosomic additions yielded fluorescent signals on the alien chromosomes only. The previously observed differences in size and brightness of the fluorescent spots were confirmed using the single alien chromosomes. FISH of PB6-4 to extended DNA fibres of the monosomic additions indicated differences in the fluorescent track lengths between the alien chromosomes. Measurements of the fluorescent tracts allowed classification into discrete groups, varying from one to three groups per B. procumbens chromosome. The data revealed that the brightness or size of the signal at mitotic metaphase and the length of the fluorescent tracks on the DNA fibres were correlated. This revised version was published online in July 2006 with corrections to the Cover Date.     

Electron microscopic investigation of mitochondrial DNA fromChenopodium album (L.)

DNA molecules from mitochondria of whole plants and a suspension culture ofChenopodium album were prepared, by a gentle method, for analysis by electron microscopy. Mitochondrial (mt) DNA preparations from both sources contained mostly linear molecules of variable sizes (with the majority of molecules ranging from 40 to 160 kb). Open circular molecules with contour lengths corresponding to 0.3–183 kb represented 23–26% of all mtDNA molecules in the preparations from the suspension culture and 13–15% in the preparations from whole plants. More than 90% of the circular DNA was smaller than 30 kb. Virtually no size classes of the mtDNA molecules could be identified, and circular or linear molecules of the genome size (about 270 kb) were not observed. In contrast, plastid (pt) DNA preparations from the suspension culture contained linear and circular molecules falling into size classes corresponding to monomers, dimers and trimers of the chromosome. About 23% of the ptDNA molecules were circular. DNA preparations from mitochondria contained a higher percentage of more complex molecules (rosette-like structures, catenate-like molecules) than preparations of ptDNA. Sigma-like molecules (putative intermediates of rollingcircle replication) were observed in mtDNA preparations from the suspension culture (18% of the circles), and in much lower amount (1%) in preparations from whole plants. The results are compared with data obtained previously by pulsed-field gel electrophoresis and discussed in relation to the structural organization and replication of the mt genome of higher plants.     

Oxidonitrergic and antioxidant effects of a low molecular weight peptide fraction from hardened bean (Phaseolus vulgaris) on endothelium

About 3000 tons of beans are not used in human food due to hardening. Several studies on bean-derived bioactive peptides have shown potential to treat some diseases, including those relying on oxidative dysfunctions. We assessed the effects of peptides extracted from hardened bean Phaseolus vulgaris (PV) on reactive oxygen species (ROS) and nitric oxide (NO) production, cytotoxic and cytoprotective effects in endothelial cells, and oxidonitrergic-dependent vasodilating effects. Extract was composed by peptide fraction <3 kDa (PV3) from hardened common bean residue. PV3 sequences were obtained and analyzed with bioinformatics. Human umbilical vein endothelial cells were treated with 10, 20, 30, and 250 µg/mL PV3. Oxidative stress was provoked by 3% H₂O₂. Cytotoxicity and cytoprotective effects were evaluated by MTT assay, whereas, ROS and NO were quantified using DHE and DAF-FM fluorescent probes by confocal microscopy. NO- and endothelium-dependent vasodilating effects of PV3 were assessed in isolated aortic rings. We found 35 peptides with an average mass of 1.14 kDa. There were no cell deaths with 10 and 20 μg/mL PV3. PV3 at 30 μg/mL increased cell viability, while cytotoxicity was observed only with 250 μg/mL PV3. PV3 at 10 μg/mL was able to protect cells from oxidative stress. PV3 also increased NO release without causing cell death. It also reduced relative ROS production induced by H₂O₂. PV3 vasodilating effects relied on endothelium-dependent NO release. PV3 obtained from low-commercial-value bean displays little cytotoxicity and exerts antioxidant effects, whereas it increases endothelial NO release.     

Nucleotide sequence and molecular characterization of plasmid-like DNAs from mitochondria of cytoplasmic male-sterile rice

Summary Two plasmid-like DNAs, B2 and B3, were isolated from mitochondria of the cytoplasmic malesterile rice, A-58 CMS. Molecular clones having their complete sequences were constructed and used as probes of mitochondrial and nuclear genomes by Southern hybridization. No evidence was found that integrated copies of either one exist in the main mitochondrial genome, but sequences homologous to both were present in the nuclear genome. The complete nucleotide sequences of B2 and B3 were established and compared to those of rice B1 and B4 and to the 1.9-and the 1.4-kbp plasmid-like DNAs of maize. Many of the sequences were common to both plant species.     

Projections from fetal neocortical transplants placed in the frontal neocortex of newborn rats. A Phaseolus vulgaris-leucoagglutinin tracing study

Summary Fetal rat neocortex grafted into lesion cavities made in the newborn rat neocortex can exchange multiple axonal connections with the host brain. Most previous studies demonstrating efferent transplant-tohost brain connections have used fluorescent retrograde tracers injected into the host brain (Castro et al. 1985, 1987; Floeter and Jones 1984; O'Leary and Stanfield 1989). Other studies have used anterograde axonal tracing with either tritium-labelled amino acids impregnating the transplant and its efferents (Floeter and Jones 1985) or horseradish peroxidase injected into the transplants (Chang et al. 1984, 1986). In the present study we used the anterograde axonal tracer Phaseolus vulgaris — leucoagglutinin (PHA-L) to examine in detail the course and termination of the efferent neocortical graft fibers. Twenty-six newborn rats had the right frontal cortex forepaw area removed by vacuum aspiration, while anesthetized by hypothermia. A piece of fetal frontal cortex 14–16 embryonic days old (E14–16) was immediately thereafter placed in the lesion, and the recipient rats allowed to survive for 5–7 months. At this time the rats were reoperated under sodium pentobarbital (Nembutal) anesthesia and the transplants iontophoretically injected with PHA-L. Two weeks later the animals were again anesthetized, perfused, and processed for PHA-L immunocytochemistry and routine histology. Analysis of acetylcholinesterase- (AChE) and Nissl-stained sections showed graft survival in 19 of the 26 animals used in this study. When these 19 brains were processed for PHA-L immunocytochemistry, 5 of them were found with certainty to have the PHA-L injection confined to the transplant. Based on these cases PHA-L-reactive fibers arising from labelled transplant neurons were traced into the ipsilateral host neocortex adjacent to the transplant and found to project through the subcortical white matter to the ipsilateral parietal neocortical area 1, and claustrum. Callosal fibers were traced to the contralateral frontal neocortical forelimb and parietal areas. Transplant fibers were also observed to descend through the caudate putamen in the dispersed fiber bundles of the internal capsule to distribute as terminal branches and varicose fibers within the mesencephalic periaqueductal gray, red nucleus, deep mesencephalic nucleus, and intermediate gray of the superior colliculus, as well as in the pontine gray. Similar fibers and terminations were present in the caudate putamen, the reticular, ventrobasal, centrolateral, posterior, and parafascicular thalamic nuclei. On the side contralateral to the transplant, fewer fibers were observed in the caudate putamen, the ventrobasal, centrolateral, and posterior thalamic nuclei, as well as more caudally in the deep mesencephalic nucleus and the intermediate gray of the superior colliculus. Our findings demonstrate that homotopic grafts of fetal rat frontal neocortex can project to the developing host brain in a manner which for most projections corresponds to the normal rat neocortical parietal area 1–2 and forelimb area. The density of these transplant-to-host projections is, however, less than in the normal rat corticofugal pathways.