Yutaka Miyazawa

The complete DNA sequence of the mitochondrial genome of Physarum polycephalum

Abstract. The complete sequence of the mitochondrial DNA (mtDNA) of the true slime mold Physarum polycephalum has been determined. The mtDNA is a circular 62,862-bp molecule with an A+T content of 74.1%. A search with the program BLAST X identified the protein-coding regions. The mitochondrial genome of P. polycephalum was predicted to contain genes coding for 12 known proteins [for three cytochrome c oxidase subunits, apocytochrome b, two F1Fo-ATPase subunits, five NADH dehydrogenase (nad) subunits, and one ribosomal protein], two rRNA genes, and five tRNA genes. However, the predicted ORFs are not all in the same frame, because mitochondrial RNA in P. polycephalum undergoes RNA editing to produce functional RNAs. The nucleotide sequence of an nad7 cDNA showed that 51 nucleotides were inserted at 46 sites in the mRNA. No guide RNA-like sequences were observed in the mtDNA of P. polycephalum. Comparison with reported Physarum mtDNA sequences suggested that sites of RNA editing vary among strains. In the Physarum mtDNA, 20 ORFs of over 300 nucleotides were found and ORFs 14–19 are transcribed.

Relationship between presence of a mother cell wall and speciation in the unicellular microalga Nannochloris (Chlorophyta)

The cell division mechanisms of seven strains from six species of Nannochloris Naumann were analyzed and compared with those of three species of Chlorella Beijerinck and Trebouxia erici Ahmadjian using differential interference microscopy and fluorescence microscopy. Nannochloris bacillaris Naumann divides by binary fission and N. coccoides Naumann divides by budding. Distinct triangular spaces or mother cell walls were found in the dividing autosporangia of the other five strains from four species of Nannochloris, three species of Chlorella, and T. erici. In an attempt to infer an evolutionary relationship between nonautosporic and autosporic species of Nannochloris, we constructed a phylogenetic tree of the actin genes using seven strains from six species of Nannochloris, three species of Chlorella, and T. erici. Nannochloris species were polyphyletic in the Trebouxiophyceae group. Two nonautosporic species of N. bacillaris and N. coccoides were monophyletic and positioned distally. Moreover, to determine their phylogenetic position within the Trebouxiophyceae, we constructed phylogenetic tree of 18S rRNA genes adding other species of Trebouxiophyceae. Nannochloris species were polyphyletic in the Trebouxiophyceae and appeared in two different lineages, a Chlorella–Nannochloris group and a Trebouxia–Choricystis group. The nonautosporic species, N. bacillaris and N. coccoides, and three autosporic species of Nannochloris belonged to the Chlorella–Nannochloris group. Nannochloris bacillaris and N. coccoides were also monophyletic and positioned distally in the phylogenetic tree of 18S rRNA genes. These results suggest that autosporulation is the ancestral mode of cell division in Nannochloris and that nonautosporulative mechanisms, such as binary fission and budding, evolved secondarily.

Comparative analysis of DNA synthesis activity in plastid-nuclei and mitochondrial-nuclei simultaneously isolated from cultured tobacco cells

Abstract Plastid-nuclei (plastid-nucleoids) and mitochondrial-nuclei (mitochondrial-nucleoids) were simultaneously isolated from cultured tobacco cells (Nicotiana tabacum L., line BY-2), and their activity synthesizing DNA in vitro was examined. The isolated plastid- and mitochondrial-nuclei incorporated about 20 and 50 pmol of dCTP, respectively, into DNA per microgram of template DNA during a 60-min incubation. The DNA synthetic activity of the two organelle-nuclei exhibited similar responses to various inhibitors; it was resistant to aphidicolin and sensitive to N-ethylmaleimide and high concentrations of ddCTP, which are all characteristics of γ-like DNA polymerase. The responses of the DNA synthetic activity in the two organelle-nuclei to pH and divalent- and monovalent-metal cation concentrations were also similar. Moreover, an in situ DNA polymerase assay following SDS-polyacrylamide gel electrophoresis revealed that DNA polymerases with an apparent molecular mass of 116 kDa were present in both the isolated plastid- and mitochondrial-nuclei, and that the two 116-kDa DNA polymerases were quite similar in terms of their sensitivity to various inhibitors, optimum assay conditions, and template preferences. These results indicate that DNA replication in plastids and mitochondria may be conducted by DNA polymerases that have quite similar characteristics.

Simultaneous isolation of cell-nuclei, plastid-nuclei and mitochondrial-nuclei from cultured tobacco cells; comparative analysis of their transcriptional activities in vitro

Abstract A procedure was established for simultaneously isolating cell-nuclei, plastid-nuclei (plastid-nucleoids) and mitochondrial-nuclei (mitochondrial-nucleoids) from cultured tobacco cells ( Nicotiana tabacum L., line BY-2). Quantitative Southern hybridization analysis revealed cross-contamination to be minimal (less than 1% in most cases). The isolated cell-, plastid- and mitochondrial-nuclei incorporated about 17, 15 and 7-pmoles of UTP, respectively, into RNA per microgram of DNA during a 60 min incubation under appropriate conditions. The isolated cell-, plastid- and mitochondrial-nuclei exhibited different sensitivities to various transcriptional inhibitors, and synthesized RNAs that are selectively hybridizable to their own DNA. The three isolated nuclei also incorporated [ γ - 32 P]ATP radioactivity into the DEAE-paper bound fraction: Although most of this incorporation was due to protein phosphorylation, a small fraction of the incorporated radioactivities was recovered as RNA, suggesting that de novo transcription initiation may occur in our assay system. The simultaneous isolation of these three nuclei and the assay of their transcriptional activities in vitro were useful for comparative analysis of their transcription apparatus. This system may also be a useful means of analyzing the transcriptional states of cell nuclear-, plastid- and mitochondrial-genomes simultaneously.

Distribution of interstitial telomere-like repeats and their adjacent sequences in a dioecious plant, Silene latifolia

Abstract. The dioecious plant Silene latifolia has large, heteromorphic X and Y sex chromosomes that are thought to be derived from rearrangements of autosomes. To reveal the origin of the sex chromosomes in S. latifolia, we isolated and characterized telomere-homologous sequences from intra-chromosomal regions (interstitial telomere-like repeats; ITRs) and ITR-adjacent sequences (IASs). Nine genomic DNA fragments with degenerate 84- to 175-bp ITRs were isolated from a genomic library and total genome of male plants. Comparing the nucleotide sequences, the IASs of the nine ITRs were classified into seven elements (IAS-a, IAS-b, IAS-c, IAS-d, IAS-e, IAS-f, and IAS-g) by sequence similarity. The ITRs were grouped into two classes (class-I and -II ITRs) according to the classification of IASs. The class-I ITRs were sub-grouped into three subclasses (subclasses-IA, -IB, and -IC ITRs) based on the arrangement of IAS elements. By contrast, the class-II ITR was located between two different IASs (IAS-f and IAS-g). Genomic Southern analyses showed that both the male and female genomes contained six (IAS-f) to 153 (IAS-d) copies of each IAS per haploid genome. Fluorescence in situ hybridization analyses showed that one IAS element, IAS-d, was distributed in the interstitial and proximal regions of the sex chromosomes of S. latifolia. The distribution of IAS-d is important evidence for past telomere-mediated chromosome rearrangements during the evolution of the sex chromosomes of S. latifolia.

Amyloplast formation in cultured tobacco cells. III Determination of the timing of gene expression necessary for starch accumulation

Abstract When BY-2 cultured tobacco (Nicotiana tabacum L.) cells were transferred to auxin-depleted culture medium containing cytokinin (benzyladenine, 1 mg/l), the starch content per cell started increasing from 18 h of culture and amyloplasts had formed by 48 h. Pulse-treatment of the cells with actinomycin D and cycloheximide for the first 12 h (or longer) of culture significantly decreased the cellular starch content after 48 h, whereas the starch content did not decrease significantly when the cells were released from the inhibition within 6 h. This suggests that nuclear gene expression necessary for amyloplast formation begins 6–12 h after the transfer. Immunoblotting analysis of the accumulation of ADP-glucose starch glycosyl transferase (starch synthase) supported this inference.

Plastid Gene Expression during Amyloplast Formation in Cultured Tobacco Cells

Summary We analyzed changes in plastid gene expression during amyloplast formation in cultured tobacco (Nicotiana tabacum L.) cells. When cultured tobacco cells (line BY 2) in the stationary phase were transferred to auxin-depleted culture medium that contained cytokinin, leucoplast-like plastids in the BY-2 cells were converted to amyloplasts within two days. The cell number, number of plastids per cell, and the relative amount of plastid DNA per cell remained nearly constant throughout this process. During amyloplast formation, transcripts for most plastid genes increased only transiently, and stopped increasing during the late phase of amyloplast development, while transcript levels for psbA (a gene for D 1 protein of photosystem 11) and psaAlB (genes for P700 apoprotein Al/A2 of photosystem 1) continued to increase. Assay of transcription in vitro using isolated plastid-nuclei (nucleoids) showed that the transcriptional activities of most plastid genes decreased dramatically during this process, whereas the transcription of psbA and psaAl B continued at a considerable rate, showing a correlation between transcription and transcript accumulation. However, the fact that increases in transcript abundance were observed for all the plastid genes investigated here in spite of the marked decrease in their transcriptional activity suggests that the degradation of transcripts may also be inactive in the amyloplasts of BY-2 cells.

Dedifferentiation of starch-storing cultured tobacco cells: effects of 2,4-dichlorophenoxy acetic acid on multiplication, starch content, organellar DNA content, and starch synthesis gene expression

Abstract. The addition of the auxin 2,4-dichlorophenoxy acetic acid (2,4-D) to tobacco Bright-Yellow-2 (BY-2) cells cultured in 2,4-D-depleted medium induced dedifferentiation of starch-storing cells into dividing cells. The changes in cell growth, starch content, organellar DNA content, and the transcript levels for the ADP-glucose pyrophosphorylase gene during the dedifferentiation of starch-storing cells were examined. Cell proliferation, decrease in starch content, and increase in DNA content began from 12–18xa0h after 2,4-D application. RNA-gel blot analysis showed a decrease in the ADP-glucose pyrophosphorylase mRNA levels, within 6xa0h of the addition of 2,4-D. Furthermore, experiments using aphidicolin revealed that starch degradation is linked with cell division.