Shigeyuki Kawano

Visualization of DNA in various phages (T4, χ, T7, ϕ 29) by ethidium bromide epi-fluorescent microscopy

DNA-containing areas in various phages (T4, χ, T7 and ϕ29) could be observed at the light microscopic level using ethidium bromide epi-fluorescent microscopy. The fluorescent intensity per phage was in linear proportion to the DNA content in each phage.

Variance of ploidy in mitochondrial nucleus during spherulation in Physarum polycephalum

In Physarum polycephalum, microplasmodia differentiated into spherules when cultures were aged for 8-10 days. Respiration rates of the microplasmodia decreased rapidly with ageing to a 90% decrease in oxygen consumption over 9 days. We studied this phenomena by isolating and characterizing mitochondria from microplasmodia and spherules at different stages of spherulation. Oxygen uptake by the isolated mitochondria decreased with spherulation. Morphological and biochemical analyses showed that mitochondrial differentiation to inactive state was characterized by a decrease not only in dimension but also of content (DNA, RNA and protein). Diminutive mitochondria contained small particle-shaped mitochondrial nuclei. The DNA content, measured by microscopic fluorometry, was about 1.15 and 0.58 X 10(-10) g, which corresponded to about 16 and 8 genome copies, respectively (e.g., 32 genome copies per mitochondrion at mitochondrial G1). Restriction endonuclease analysis showed that the physical structure and methylation pattern of the mtDNA had not changed although the DNA content per mitochondrion had decreased remarkably with spherulation. This showed that changes in the ploidy level of the mitochondrial nucleus during spherulation were due to reduction in the number of whole mitochondrial genomes.

Structural homogeneity of mitochondrial DNA in the mitochondrial nucleoid of Physarum polycephalum

Mitochondrial DNA (mtDNA) of Physarum polycephalum was isolated gently by CsCl centrifugation. The mtDNA was linear with molecular weights ranging from 25 . 10(6) to 45 . 10(6) and heterogeneous in size. Nevertheless, thermal transition profiles of the mtDNA suggested that this DNA fraction was more homogeneous than nuclear DNA. Exhaustive digestions of this DNA with restriction endonucleases yielded unique fragments, and then the total of their molecular weights of each digest was around 45 . 10(6). This value is equivalent to the maximum molecular weight estimated using electron microscopy and electrophoresis. Moreover, EcoRI digests of the mtDNA fractionated by the sucrose gradient showed unequimolar quantities of large fragments and a high background between bands. These results suggest that the mtDNA of Physarum has a homogeneous base sequence, and that the size heterogeneity of the mtDNA is attributable to degradation of the DNA under isolation procedures. The mtDNA was cleaved by EcoRI and XhoI to yield 16 and 7 fragments, respectively. A physical map of these fragments was constructed using the routine mapping procedures. The physical map showed that the mitochondrial genome of Physarum was linear with molecular weight of 45 . 10(6). We concluded therefore that the mitochondrial nucleoid is a structure in which the homogeneous mtDNA is highly amplified.

Amyloplast nucleoids in sycamore cells and presence in amyloplast DNA of homologous sequences to chloroplast genes

Employing specific dye-stain followed by fluorescence microscopy, evidence was obtained for the presence of amyloplast nucleoids in protoplasts as well as in the amyloplasts isolated from the suspension- cultured cells of sycamore (Acer psuedoplatanus L.), which are devoid of chlorophylls and grow heterotrophically. We have isolated and partially characterized amyloplast DNA. The presence of six homologous sequences to chloroplast genes encoding (i) apoprotein of P700, (ii) 32 kDa protein (photogene 32), (iii) alpha subunit of CF1, (iv) large subunit of RuBisCO, (v) beta and epsilon subunits of CF1 and (vi) 16S ribosomal RNA, has been revealed by hybridization experiments, using gene probes from maize chloroplast DNA.

Isolation and characterization of a membrane-DNA complex in the mitochondria of Physarum polycephalum☆

A membrane-DNA complex was isolated by centrifugation of sheared lysate of isolated mitochondria in 20-60% sucrose step solution. Analyses using Hoechst 33258/CsCl density gradient centrifugation and restriction endonuclease treatment showed that DNA in the membrane-DNA complex was AT-rich compared with total mitochondrial DNA (mt DNA) and contained Eco RI fragments of E-4, 5 and 8, which were localized on the right hand of Physarum mitochondrial genome. Phenethyl alcohol (PEA) and ethidium bromide (EB) could disrupt the membrane-DNA complex to release DNA fragments from their complex in vitro. Addition of 0.5% or more PEA, which released 80-90% of the DNA from the membrane-DNA complex in vitro, inhibited not only mitochondrial nuclear division but also mitochondrial division in vivo. EB treatment at more than 1 mg/ml disrupted the membrane-DNA complex in vitro to release 77% of the total DNA in the complex. Addition of 10 micrograms/ml EB induced unequal mitochondrial nuclear division in the microplasmodia, e.g., a dividing dumbbell-shaped mitochondrion had the mt-nucleus in one side and as a result formed then one nucleated and one enucleated mitochondrion. From the EB-pretreated mitochondria, a lesser amount of the membrane-DNA complex was isolated than from the control. These findings mean than the unequal mt-nuclear division is due to dissociation of DNA and the membrane system in the membrane-DNA complex. They strongly suggested that the DNA region (E-4, 5 and 8), where the mitochondrial nucleus is associated with the mitochondrial membrane system plays an important role in mitochondrial nuclear division.