Studies on the behavior of organelles and their nucleoids in the root apical meristem of Arabidopsis thaliana (L.) Col.

The behavior of cell nuclei, mitochondrial nucleoids (mt-nucleoids) and plastid nucleoids (ptnucleoids) was studied in the root apical meristem of Arabidopsis thaliana. Samples were embedded in Technovit 7100 resin, cut into thin sections and stained with 4′-6-diamidino-2-phenylindole for light-microscopic autoradiography and microphotometry. Synthesis of cell nuclear DNA and cell division were both active in the root apical meristem between 0 μm and 300 μm from the central cells. It is estimated that the cells generated in the lower part of the root apical meristem enter the elongation zone after at least four divisions. Throughout the entire meristematic zone, individual cells had mitochondria which contained 1–5 mt-nucleoids. The number of mitochondria increased gradually from 65 to 200 in the meristem of the central cylinder. Therefore, throughout the meristem, individual mitochondria divided either once or twice per mitotic cycle. By contrast, based on the incorporation of [³H]thymidine into organelle nucleoids, syntheses of mitochondrial DNA (mtDNA) and plastid DNA (ptDNA) occurred independently of the mitotic cycle and mainly in a restricted region (i.e., the lower part of the root apical meristem). Fluorimetry, using a videointensified microscope photon-counting system, revealed that the amount of mtDNA per mt-nucleoid in the cells in the lower part of the meristem, where mtDNA synthesis was active, corresponded to more than 1 Mbp. By contrast, in the meristematic cells just below the elongation zone of the root tip, the amount of mtDNA per mt-nucleoid fell to approximately 170 kbp. These findings strongly indicate that the amount of mtDNA per mitochondrion, which has been synthesized in the lower part of the meristem, is gradually reduced as a result of continual mitochondrial divisions during low levels of mtDNA synthesis. This phenomenon would explain why differentiated cells in the elongation zone have mitochondria that contain only extremely small amounts of mtDNA. This work was supported by a Grant-in Aid (T.K.) for Special Research on Priority Areas (Project No. 02242102, Cellular and Molecular Basis for Reproduction Processes in Plants) from the Ministry of Education, Science and Culture of Japan and by a Grant-in Aid (T.K.) for Original and Creative Research Project on Biotechnology from the Research Council, Ministry of Agriculture, Forestry and Fisheries of Japan.     

The course of chloroplast DNA replication and its relationship to other reproductive processes in the chloroplast and nucleocytoplasmic compartment during the cell cycle of the algaScenedesmus quadricauda

Summary DNA containing structures (cellular, chloroplast and mitochondrial nuclei) were stained with the fluorochrome DAPI. Fluorescence intensity, as a measure of DNA content, was estimated during the mitotic cycle in synchronized populations of the chlorococcal alga,Scenedesmus quadricauda. In cells yielding eight daughter cells, three consecutive steps in chloroplast DNA increase occurred over one mitotic cycle. The first step was performed shortly after releasing the daughter cells, the second and third steps occurred consecutively during the first half of the mitotic cycle. Commitment to chloroplast DNA replication was chronologically separated from commitment to division of chloroplast nuclei, revealing that these two chloroplast reproductive steps were under different control mechanisms. The replication of chloroplast DNA occurred at a different time to that of cell-nuclear DNA. The coordination of chloroplast reproductive processes and those in the nucleocytoplasmic compartment were governed by the mutual trophic and metabolic dependency of these compartments rather than by any direct or feedback control controlled by either of them.Abbreviations DAPI 46-diamidino-2-phenylindole - ptDNA DNA in chloroplast nuclei - nucDNA DNA in cell nuclei     

SYNCHRONIZATION OF CHLOROPLAST DIVISION IN THE ULTRAMICROALGA CYANIDIOSCHYZON MEROLAE (RHODOPHYTA) BY TREATMENT WITH LIGHT AND APHIDICOLIN

A system of highly synchronized chloroplast divisions was developed in the unicellular red alga Cyanidioschyzon merolae De Luca, Taddei, & Varano. Chloroplast divisions were examined by epifluorescence microscopy following treatments with light and inhibitors. When the cells during stationary phase were transferred into a new medium under a 12:12 h LD cycle, chloroplasts, mitochondria, and cell nuclei divided synchronously in that order soon after the initiation of dark periods. More than 40% of the cells contained dividing chloroplasts. To obtain a system of highly synchronized cell division and chloroplast division, the cells synchronized by a 12:12 h LD cycle were treated with various inhibitors. Nocodazole and propyzamide did not affect cell and organelle divisions, whereas aphidicolin markedly inhibited cell-nuclear divisions and cytokinesis and induced a delay in chloroplast division. More than 80% of the cells contained dividing chloroplasts when cells synchronized by light were treated with aphidicolin for 12 h. This synchronized system will be useful for studies of the molecular and cellular mechanisms of organelle divisions .     

Analysis of a plastid gene cluster reveals a close relationship betweenCyanidioschyzon andCyanidium

Cyanidioschyzon merolae andCyanidium caldarium are representative species among of the most primitive algae, although the two species are distinctly different in various morphological traits. We determined the nucleotide sequence of therbcL gene and a flanking 8-kb region in the plastid genome of each of these algae. In both algae, 12 genes were identified in this region, in an identical order. This gene order is not conserved in the plastid genomes of other species of the kingdom Plantae that have been sequenced to data. An additional unidentified open reading frame was also found in the two algae that we analyzed, which has not been described in any other species of algae includingPorphyra purpurea. Comparison of the amino acid sequences of selected genes also supported the conclusion thatCyanidioschyzon merolae andCyanidium caldarium are closely related and that they are distinct from other rhodophytes. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession numbers D63675 and D63676.     

Protein which interacts with a stimulatory factor of RNA polymerase II of Ehrlich ascites tumor cells

When partially purified Ehrlich ascites tumor RNA polymerase II was further purified on a column of phosphocellulose, stimulation of its catalysis of RNA synthesis by stimulatory factor S-II was greatly decreased. This decrease in sensitivity to the stimulatory factor was reversible: the enzyme eluted from phosphocellulose became sensitive to the factor when mixed with a protein fraction eluted from the phosphocellulose at high salt concentration. Evidence was obtained that this protein, named helper protein, binds, to the enzyme eluted from phosphocellulose, causing it to recover sensitivity to stimulatory factor S-II.     

Behavior of organelle nuclei (nucleoids) in generative and vegetative cells during maturation of pollen inLilium longiflorum andPelargonium zonale

Summary The behavior of organelle nuclei during maturation of the male gametes ofLilium longiflorum andPelargonium zonale was examined by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and Southern hybridization. The organelle nuclei in both generative and vegetative cells inL. longiflorum were preferentially degraded during the maturation of the male gametes. In the mature pollen grains ofL. longiflorum, there were absolutely no organelle nuclei visible in the cytoplasm of the generative cells. In the vegetative cells, almost all the organelle nuclei were degraded. However, in contrast to the situation in generative cells, the last vestiges of organelle nuclei in vegetative cells did not disappear completely. They remained in evidence in the vegetative cells during germination of the pollen tubes. InP. zonale, however, no evidence of degradation of organelle nuclei was ever observed. As a result, a very large number of organelle nuclei remained in the sperm cells during maturation of the pollen grains. When the total DNA isolated from the pollen or pollen tubes was analyzed by Southern hybridization with a probe that contained therbc L gene, for detection of the plastid DNA and a probe that contained thecox I gene, for detection of the mitochondrial DNA, the same results were obtained. Therefore, the maternal inheritance of the organelle genes inL. longiflorum is caused by the degradation of the organelle DNA in the generative cells while the biparental inheritance of the organelle genes inP. zonale is the result of the preservation of the organelle DNA in the generative and sperm cells. To characterize the degradation of the organelle nuclei, nucleolytic activities in mature pollen were analyzed by an in situ assay on an SDS-DNA-gel after electrophoresis. The results revealed that a 40kDa Ca²⁺-dependent nuclease and a 23 kDa Zn²⁺ -dependent nuclease were present specifically among the pollen proteins ofL. longiflorum. By contrast, no nucleolytic activity was detected in a similar analysis of pollen proteins ofP. zonale.     

Structural requirements for interruption of protein translocation across rough endoplasmic reticulum membrane

Co-translational translocation of proteins across the membrane of rough endoplasmic reticulum (ER) is interrupted by particular amino acid sequences, which are functionally termed "stop-transfer sequence." We analyzed the structural requirements for the interruption of the peptide translocation. By the manipulation of the cDNA of interleukin 2 (IL2), which passes through ER membrane co-translationally, the middle portion of the IL2 molecule was replaced with systematically altered hydrophobic segments, leucine, alanine, or leucine/alanine mixed clusters. Furthermore, charged amino acid residues were introduced just downstream of the hydrophobic segments. These modified IL2 peptides were synthesized with wheat germ cell-free system in the presence of rough microsomes and the topology of the peptides in the microsomes was assessed by post-translational digestion with proteinase K. We obtained the following results. (i) Each modified protein was processed to the mature form but the extent of stop-translocation varied widely. The ratio of the stopped to the translocated products increased as the length and hydrophobicity of the inserted segment increased. (ii) Shorter hydrophobic segments than naturally occurring native transmembrane segment promoted stop-translocation. (iii) Proteins with hydrophobic segments followed by positive charges were more efficiently stop-translocated than those having negative charges. (iv) If the hydrophobicity of the segment was sufficiently high, the positive charges after the segment were not essential for stop-translocation. We also suggest that the stop-transfer process includes protein-protein interaction between the hydrophobic segment and translocation channel.     

Identification of storage-protein messenger RNA of the fleshfly Sarcophaga peregrina.

Storage-protein mRNA was found to be abundant in poly(A)-containing RNA extracted from the fat-body of third-instar larvae of Sarcophaga peregrina (fleshfly). This RNA sedimented at the position of 19S on sucrose-density-gradient centrifugation and the product of its translation in vitro was 75K protein (protein of mol.wt. 75 000), which was precipitated specifically with antibody against storage protein. This product was suggested to contain a signal sequence that is missing in mature storage protein. The poly(A)-containing RNA was also found to contain much of another mRNA coding for 25K protein (protein of mol.wt. 25 000), but the function of this protein is unknown.     

Variance of ploidy in Candida albicans

Determination of ploidy was performed on isolates of Candida albicans from clinical sources by measuring nuclear DNA content with fluorescent microscope photometry. By this criterion and UV irradiation survival experiments, haploid, diploid, and tetraploid strains were identified in this organism. The dimensions of nucleus-associated organelles (equivalent to spindle pole bodies) in these strains increased as a function of ploidy number.