Yukiko Matsuzawa

Collapse of single DNA molecule in poly(ethylene glycol) solutions

The compactization of a single DNA molecule in polyethylene glycol (PEG) solution was investigated both theoretically and experimentally. A theory is proposed taking into account the polyelectrolyte effect and redistribution of PEG within DNA coils. This approach makes it possible to describe the dependence of critical value, c, of PEG concentration at the point of DNA collapse on the degree of PEG polymerization, P, and on the concentration of low‐molecular salt, ns. Observation of single DNA molecule in solution of PEG has been carried out by means of fluorescence microscopy which allows one to observe the conformation of individual DNA directly. Direct evidence that the coil–globule transition of DNA occurs as first order phase transition was obtained. It was confirmed that the critical concentration of PEG decreases with an increase of the degree of PEG polymerization and salt concentration. The width of the coexistence region of coil and globule was found to be dependent on salt concentration and deg...

Acidisphaera rubrifaciens gen. nov., sp. nov., an aerobic bacteriochlorophyll-containing bacterium isolated from acidic environments.

Four strains of aerobic, mesophilic, acidophilic bacteria that produced bacteriochlorophyll (BChl) a were isolated from acidic hot springs and mine drainage. The characteristics of the four isolates were almost identical. The isolates were strictly aerobic and chemo-organotrophic. They were gram-negative, non-motile cocci and coccobacilli, formed salmon-pink colonies on solidified media and produced BChl a and carotenoids only under aerobic growth conditions. The cells also produced small amounts of zinc-substituted BChl a when grown in the presence of 1 mM zinc sulfate. Anaerobic growth in the light was not found, but aerobic growth was stimulated by continuous incandescent illumination. The isolates grew in a pH range of 3.5-6.0, with pH optima of 4.5-5.0. A phylogenetic analysis based on 16S rDNA sequences showed that the isolates clustered in the major acidophilic group of the class Proteobacteria, which includes species of the genera Acidiphilium and Rhodopila. The anaerobic phototrophic bacterium Rhodopila globiformis was the closest relative to the new isolates (95% level of sequence similarity). The G+C content of the genomic DNA of the isolates was 69.1-69.8 mol%. On the basis of these results, it was concluded that the four isolates should be classified into a new genus and a new species, for which the name Acidisphaera rubrifaciens is proposed. The type strain is strain HS-AP3T (= JCM 10600T).

Direct observation of the biphasic conformational change of DNA induced by cationic polymers

The interaction between T4 DNA and basic polypeptides was observed using fluorescence microscopy. Free DNA molecules exhibited random Brownian motion accompanying the conformational change. With the addition of polycation, such as histone and polyarginine. DNA molecules tended to shrink to become spherical shapes. The persistent lengths and the distributions of long axis lengths of DNA‐polyarginine complexes were determined from the video images at various polyarginine concentrations. It is demonstrated that the conformation of DNA changes in a biphasic manner in the presence of polyarginine.

Discrete phase transition of giant DNA dynamics of globule formation from a single molecular chain

Abstract We found that a single chain of double strand DNA molecule undergoes a first-order phase transition by the direct observation of the conformation in individual DNA with fluorescence microscopy. It has become clear that a single DNA chain changes its higher order structure between expanded coil and shrunken globule in a discrete manner. The effective volume occupied by single DNA chain in the coil state is ca. 10 4 times greater than that in the globule state. The dynamic process of the globule formation, or nucleation and growth, is analyzed by measuring individual DNA molecules with fluorescence microscopy. We show that the apparent contour length of the DNA coil decreases with a constant speed, accompanied by the growth of the globular structure.

Change of the higher order structure in a giant DNA induced by 4',6-diamidino-2-phenylindole as a minor groove binder and ethidium bromide as an intercalator

Abstract From a quantitative study of the contour length and the persistent length of DNA molecules with fluorescence microscopy, it is shown that 4′, 6-diamidino-2-phenylindole (DAPI) and ethidium bromide (EB) change the higher order structure of DNA in a different manner.

Manipulation of single coiled DNA molecules by laser clustering of microparticles

We have developed a method of manipulating single DNA molecules for application in single-molecule analysis. Using a bead cluster formed by laser trapping, the technique allows single DNA molecules to be manipulated at any point on the molecule without the need for prior chemical modification as in DNA-bead complex techniques. We describe the method and the characteristics of cluster formation, and present examples of actual DNA molecule manipulation.

Manipulation of globular DNA molecules for sizing and separation.

Handling large DNA molecules, such as chromosomal DNA, has become necessary due to recent developments in genome science. However, large DNA molecules are fragile and easily broken by shear stress accompanying flow in solution. This fragility causes difficulties in the preparation and handling of large DNA molecules. This study demonstrates the transition of DNA from a coiled to a globular form, which is highly condensed. This state suppresses DNA fragmentation due to shear stress in solution. The transition enables large DNA molecules to undergo mechanical manipulation. We confirmed that the fluorescence intensity of stained globular DNA increases with increasing length, suggesting that the resistance of globular DNA to shear stress is the factor that allows analysis of large DNA by flow cytometry.

Ultrastructure of the acidophilic aerobic photosynthetic bacterium Acidiphilium rubrum.

Abstract. The ultrastructure of cells of Acidiphilium rubrum, which is an acidophilic aerobic photosynthetic bacterium containing zinc-complexed bacteriochlorophyll a, was studied by electron microscopy with the rapid substitution technique. Thin-section electron microscopy indicated that any type of internal photosynthetic membranes was not present in this organism despite a relatively high content of the photopigment. The majority of cells had poly-β-hydroxybutyrate granules and electron-dense spherical bodies identified as being polyphosphate granules. When the organism was grown chemotrophically with 0.1% FeSO4, it produced another group of electron-dense granules that were associated with the inner part of the cytoplasmic membrane. An energy-dispersive X-ray analysis showed that these membrane-bound, electron-dense granules contained iron.

Geometric manipulation of DNA molecules with a laser

The present study reports on the geometric manipulation of DNA molecules condensed by treatment with PEG and salt with laser on a microscope. To date, the majority of previously reported laser studies have dealt with the trapping or transportation of target objects. Here, we demonstrate the successful attachment and transformation of condensed DNA molecules as well as the construction of various configurations. Laser manipulation of DNA has fruitful applicability in chemistry, life science, and biotechnology.

Manipulation of chromosomal DNA and localization of enzymatic activity

Observation of reaction among a few molecules will contribute to obtain profitably detailed information in biological fields such as molecular biology, which cannot be obtained from an in vitro system containing a large amount of molecules. This paper describes manipulation of a yeast chromosomal DNA and localization of enzymatic activity on a single or a few DNA molecules, as follows. (1) Globule structure in DNA molecules suppressed fragmentation of long DNA molecules due to sharing stress. It was demonstrated experimentally that the DNAs in globule state were tough for sharing stress. It means that globule formation enables mechanical manipulation of a long DNA molecule. We could make a successful recovery of a single globular chromosomal DNA into a glass capillary in a solution. (2) Using local area temperature control technique, activity of restriction enzyme could be activated only in a localized area by raising a local temperature. Then chromosomal DNAs were digested about 10 /spl mu/m around the laser focal spot on their DNAs. These single molecule technologies may contribute to several fields, especially biotechnology and genome analysis.