Fluorescence microscopic study of the formation of giant mitochondrial nuclei in the young ovules ofPelargonium zonale

Summary The size of mitochondrial genomes in higher plants are known to range from 200 to 2400 kilobase pairs. However, we failed to identify cytochemically any mitochondria that contain an identifiable master mitochondrial genome. In the present experiments, we have found the giant mitochondrial nuclei which have the capacity for including the master mitochondrial genome in the young ovaries ofPelargonium zonale by use of a 4-6-diamidino-2-phenylindole (DAPI) epifluorescence microscopy, a Technovit embedding, and a video-intensified photon counting system.     

Galactosylceramide- and lactosylceramide-loading studies in cultured fibroblasts from normal individuals and patients with globoid cell leukodystrophy (Krabbe's disease) and GM1-gangliosidosis

The metabolism of galactosylceramide and lactosylceramide in cultured fibroblasts was studied using the lipid-loading test. These compounds were incorporated into the fibroblasts yet only small amounts of the incorporated lipids were hydrolyzed unless additional phospholipid was mixed with the glycolipid before loading. Among phospholipids, phosphatidylserine was the most effective for incorporation and hydrolysis of the glycolipids, while phosphatidylcholine inhibited the incorporation of the glycolipids. Using filtration techniques, light scattering analyses and subcellular fractionation, the particle size of glycolipid in the culture medium was found to be critically important for the incorporation of the lipids into the cells and their transportation to the lysosomes. The particle sizes of the glycolipids were decreased by mixing with phosphatidylserine. Furthermore, the negative charge in phosphatidylserine may be necessary for the glycolipid transportation into the lysosomes. In fibroblasts from patients with globoid cell leukodystrophy, 40-50% of galactosylceramide was hydrolyzed on the 4th day of culture, a time when the control fibroblasts had hydrolyzed it about 80%. This finding is in contrast with observations made on fibroblasts with other sphingolipidoses which showed near-zero degradation in corresponding substrate-loading tests. In fibroblasts from patients with either globoid cell leukodystrophy of GM1-gangliosidosis, hydrolysis of lactosylceramide was fairly normal yet somewhat lower than control values on any day of culture, thereby indicating that, in the loading tests, lactosylceramide seems to be hydrolyzed with similar levels of enzyme activities by two distinct beta-galactosidases, galactosylceramidase and GM1-ganglioside beta-galactosidase.     

Optical isolation of individual mitochondria ofPhysarum polycephalum for PCR analysis

Summary We attempted to amplify a specific region of mitochondrial DNA (mtDNA) using the polymerase chain reaction (PCR) from fewer than ten mitochondria isolated individually by microdissection or use of an optical tweezer. We selected preliminarily isolated mitochondria fromPhysarum polycephalum as the model materials and tried to amplify the mtDNA region corresponding to the specific mitochondrial plasmid of this true slime mould. For separation of a few mitochondria from the mitochondrial population, we initially used a destruction method in which excluded mitochondria were disrupted by a UV laser. However, mtDNA was still amplified, although weakly, from mitochondria that had been destroyed by the UV laser. Therefore, we used an optical tweezer to trap individual mitochondria and separate them from the others. The required number of mitochondria were separated from the mitochondrial suspension through a narrow canal of isolation buffer and used directly for PCR amplification. The results showed that the mtDNA could be amplified from at least 9 mitochondria trapped by the optical tweezer.Abbreviations DAPI 4,6-diamidino-2-phenylindole - EDTA ethylenediaminetetraacetic acid - mtDNA mitochondrial DNA - PCR polymerase chain reaction     

PHYLOGENETIC ANALYSIS OF MORPHOLOGICAL SPECIES OF CARTERIA (VOLVOCALES,CHLOROPHYTA) BASED ON rbcL GENE SEQUENCES1

Four related species in the unicellular volvocalean genus Carteria [C. crucifera Pascher, C. eugametos Mitra, C. inversa (Korshikov) Bourrelly and C. cerasiformis Nozaki et al.] were delineated on the basis of recent comparative light and electron microscopy of a large number of culture strains. However, the species thus delineated may not represent natural or monophyletic entities. In the present study, 1128 base pairs of the chloroplast protein-coding gene (large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase gene) from 12 Carteria strains representing the four species as well as from related volvocalean species were analyzed to elucidate the phylogenetic status of the taxonomic or morphologic species of Carteria. The sequence data showed that the 12 Carteria strains exhibit four robust monophyletic groups which are strictly consistent with the four taxonomic species. These results are discussed in relation to contrasting results found in other microalgal genera. It is concluded that phylogenetic analysis, based on DMA sequence data and comparative morphologic characterization of species and using a large number of culture strains, is essential to a natural system of microalgal species taxonomy.     

Checkpoint control on mitochondrial division inCyanidioschyzon merolae

Summary It is generally accepted that mitochondria proliferate by division. However, since the apparatus for mitochondrial division was discovered only recently, the basic mechanism of mitochondrial division remains poorly understood. The unicellular red algaCyanidioschyzon merolae is the only organism in which the existence of the apparatus for mitochondrial division (mitochondrion-dividing ring) has been proved by electron microscopy. Since mitochondrial division, mitosis, and cytokinesis regularly occurred in that order, we can assume that tight linkage exists between mitochondrial division and the mitotic cycle. To examine this assumption, we performed experiments with aphidicolin, a specific inhibitor of DNA polymerase , using cells that had been synchronized by a 12 h light/12 h dark treatment. The effects of aphidicolin onC. merolae cells were examined by both epifluorescence and electron microscopy. When cells synchronized at the S phase were treated with aphidicolin, neither mitosis nor cytokinesis occurred. Epifluorescence microscopy after staining with 3,3-dihexyloxacarbocyanine iodide (DiOC₆; a mitochondrion-specific fluorochrome) revealed that mitochondrial division was also completely inhibited. Nevertheless, electron-microscopic examination of the aphidicolin-treated cells clearly revealed the presence of a mitochondrion-dividing ring in mitochondria in all cells examined, in spite of the absence of mitochondrial division. Microbodies, which might be related to mitochondrial division inC. merolae, also failed to divide and became attached to the mitochondrion-dividing rings. These results imply the presence of a checkpoint control mechanism that inhibits division of mitochondria and microbodies in the absence of the synthesis of cell-nuclear DNA.Abbreviation DiOC₆ 3,3-dihexyloxacarbocyanine iodide     

Isolation of organellar DNA from Codium fragile (Codiaceae, Codiales, Ulvophyceae)

Organellar DNA was isolated from Codium fragile (Suringar) Hariot (Codiaceae, Codiales, Ulvophyceae) by CsCI-buoyant density centrifugation in the presence of Hoechst dye 33258. Three bands were formed by ultracentrifugation and each fraction of DNA was identified by Southern hybridization. The uppermost fraction was identified as chloroplast DNA, the middle fraction was nuclear DNA and the bottom fraction was mitochondrial DNA. Nuclear rDNA was isolated in the same fraction as mitochondrial DNA. The estimated genome size of mitochondrial DNA by analysis with restriction endonucleases was more than 141.6 kb, which was larger than that of microalgae but smaller than land plants. Restriction endonuclease analysis of the chloroplast DNA showed no difference with that known of C. fragile in New York.     

Peg5/Neuronatin is an imprinted gene located on sub-distal chromosome 2 in the mouse.

We have established a systematic screen for imprinted genes using a subtraction-hybridization method with day 8.5 fertilized and parthenogenetic embryos. Two novel imprinted genes, Peg1/Mest and Peg3, were identified previously by this method, along with the two known imprinted genes, Igf2 and Snrpn. Recently three additional candidate imprinted genes, Peg5-7 , were detected and Peg5 is analyzed further in this study. The cDNA sequence of Peg5 is identical to Neuronatin, a gene recently reported to be expressed mainly in the brain. Two novel spliced forms were detected with some additional sequence in the middle of the known Neuronatin sequences. All alternatively spliced forms of Peg5 were expressed only from the paternal allele, confirmed using DNA polymorphism in a subinterspecific cross. Peg5/Neuronatin maps to sub-distal Chr 2, proximal to the previously established imprinted region where imprinted genes cause abnormal shape and behavior in neonates.     

A ZYGOTE-SPECIFIC PROTEIN WITH HYDROXYPROLINE-RICH GLYCOPROTEIN DOMAINS AND LECTIN-LIKE DOMAINS INVOLVED IN THE ASSEMBLY OF THE CELL WALL OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)

The cell wall of Chlamydomonas reinhardtii zygotes, which forms rapidly after the fusion of wall-free gametes, provides a tractable system for studying the properties and assembly of hydroxyproline-rich glycoproteins, the major proteinaceous components of green algal and plant cell walls. We report the cloning of the zsp2 gene and the analysis of its ZSP-2 product, a 58.9 kDa polypeptide that is synthesized exclusively by zygotes. The protein contains two (SP)_x repeats, establishing it as a member of the cell wall hydroxyproline-rich glycoproteins family. It also contains a 4-fold iteration of an amino acid sequence centered around cysteine residues, a configuration found in both plant and animal lectins. Furthermore, we report four observations on pellicle composition and production. First, cell-free preparations of the pellicle matrix are rich in hydroxyproline, arabinose, and galactose and contain bundles of very long fibrils. Second, glutathione blocks pellicle formation and results in the accumulation of long fibrils in the growth medium. Third, antibody to ZSP-2 also blocks pellicle formation. Fourth, ZSP-2 immunolocalizes to the boundary between the outer layers of the wall proper and the pellicle matrix. These observations are consistent with the possibility that the Cys-rich (glutathione-sensitive) lectin-like domains of ZSP-2 may bind to sugar residues on the long fibrils and anchor them to the cell wall, thereby initiating and maintaining pellicle formation.     

Studies on Plastid-Nuclei (Nucleoids) in Nicotiana tabacum L. II. Disassembly and Reassembly of Proplastid-Nuclei Isolated from Cultured Cells

The effects of various concentrations of NaCl on the compactstructure of proplastid-nuclei (pp-nuclei) isolated from culturedtobacco cells were examined by fluorescence microscopy usinga DNA-specific fluorochrome, 4, 6-diamidino-2-phenylindole.Simultaneously, behavior of the four proplastid DNA-bindingproteins (mol wt: 69kDa, 31 kDa, 30kDa and 14kDa) identifiedpreviously (Nemoto et al. 1988) was investigated by SDS-polyacrylamidegel electrophoresis. When the concentration of NaCl was increased, the isolated pp-nucleiwere gradually dispersed, and at concentrations greater than0.4 M NaCl they were almost completely dispersed. During thisdisassembly process, the 31-kDa and 30-kDa proteins dissociatedfrom the pelletable pp-nuclear fraction to the supernatant at0.1 M NaCl, whereas the 69-kDa and 14-kDa proteins dissociatedto the supernatant only at 0.4 M NaCl. When the concentrationof NaCl was decreased again by dialysis, the pp-nuclei whichhad been dispersed by 2 M NaCl were gradually reassembled intocompact structures which were almost identical to the originalpp-nuclei. During this reassembly process, the 69-kDa and 14-kDaproteins first returned to the pellet fraction, and subsequentlythe 31-kDa and 30-kDa proteins moved into the pellet. This behavior of the proplastid DNA-binding proteins stronglysuggests that the association of these proteins with plastid-DNAis responsible for the compact organization of pp-nuclei. Inaddition, it was indicated that the 69-kDa and 14-kDa proteinsare more tightly bound to plastid-DNA than are the 31-kDa and30-kDa proteins. ⁴Present address: Biological Laboratory, Faculty of Science,Nara Women's University, Nara, 630 Japan. (Received August 24, 1988; Accepted February 28, 1989)