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Y Husimi

Publications and source records attributed to Y Husimi.

36 records · Page 2Linked to original sources
PubMed1992

Structural analysis of nucleic acids by precise denaturing gradient gel electrophoresis: I. Methodology.

A method to convert the conventional denaturing gradient gel electrophoresis into a highly reproducible experimental system was developed. It was based on the following experimental findings; (i) dyes, which are small molecules, do not exhibit mobility changes attributed to their conformational change while nucleic acids do; and (ii) most of the mobility shifts caused by experimental fluctuations could be cancelled by normalizing the mobility of a sample with respect to the corresponding one of a dye. The method involves co-migration of internal reference dyes with samples (nucleic acids), and computer-aided data processing, allowing us to obtain the relative mobility of nucleic acids with respect to a dye throughout the denaturing gradient. The overall pattern of the relative mobilities thus obtained, named the normalized mobility profile (NMP), corresponded well to conformational changes of a macromolecule induced by denaturing effects. This method provides us with objective data without using internal macromolecular references, which not only guarantees the precision but also extends the range of application of the denaturing gradient method.

Base Sequence↗
PubMed1992

Structural analysis of nucleic acids by precise denaturing gradient gel electrophoresis: II. Applications to the analysis of subtle and drastic mobility changes of oligo- and polynucleotides.

Precise denaturing gradient gel electrophoresis was effectively applied to various kinds of oligo- and polynucleotides. The analyses on oligonucleotides revealed that every oligonucleotide has its own characteristic normalized mobility profile (NMP), which can be used to identify, characterize and classify the molecules. The precise system also enabled us to obtain unequivocally the mobility transitions corresponding to the melting of hairpin structures of oligonucleotides, single-stranded (ss) DNAs, and RNAs. Another application to co-migration and separate migration experiments demonstrated that there were significant binding interactions between two species of ss molecules of similar mobility, even when they have little complementarity with each other. When the precise temperature gradient gel electrophoresis was applied to double-stranded DNAs, it could be confirmed with high reliability that the mobility transitions observed correspond to cooperative meltings and strand dissociations. Through these experiments, mu m, a parameter defined as a mobility transition point, was shown to be effective to deal with those phenomena quantitatively.

Base Sequence↗
PubMed1989

Selection and evolution of bacteriophages in cellstat.

Objectives of this work were as follows: 1. to establish a laboratory experimental system utilizable in a biophysical approach to molecular evolution; and 2. to provide real world parameters to theories of molecular evolution, especially to Eigen's theory of quasi-species. Secretion type bacteriophage fd of E. coli, closely related phages and artificial chimera phages of fd, and a virulent phage Q beta of E. coli were cultured continuously in a specially designed fermenter called a "cellstat". A phage is cultured in a flow of host bacterial cells. Due to its high dilution rate, the mutant cell could not be selected in the cellstat. It was therefore recognized that the cellstat is suitable for study of the selection and evolution process of a bacteriophage under well-defined environmental conditions without interference from host cell mutations. Population dynamics of bacteriophages of various types in the cellstat were studied theoretically by computer simulation and experimentally. A genetically invariable pure population of phage behaves like an open non-linear chemical reaction system. An invariable mixed population shows a selection process, while a variable population generates an evolution process. Kinetic constants describing the dynamics were determined by curve fitting between the theoretical and the experimental curve obtained from competition experiments and from biological relaxation experiments. One of the most important kinetic parameters thus obtained was the selection coefficient, and its dependence on the base sequence of phage DNA. We drew a local landscape of the selection coefficient near the fd sequence on the base sequence space. From this landscape we were able to confirm the importance of slightly deleterious mutants in molecular evolution. We also confirmed the possibility of developing an evolutionary molecular engineering using a cellstat as an evolution reactor and fd phage as a working replicon. Novelties of this work were as follows: 1. the first stable continuous culture of a bacteriophage was achieved with a cellstat; 2. a local landscape of selection coefficient near the fd sequence on the sequence space was the first experimental drawing of such a map; 3. a biological relaxation method was realized to measure kinetic constants of a biological kinetic process, or molecular evolution; and 4. a practical engineering process of evolutionary molecular engineering was proposed.

Bacteriophages↗
PubMed1988

Bacterial bioassay of microgram to subnanogram quantities of glucose.

A simple and highly sensitive bioassay for the measurement of glucose is described. The method is based on the fact that the yields of cultured bacteria depend on the amount of glucose input, which is a limiting factor in Davis minimal medium. The number of cells cultured under fixed conditions was shown to be dependent on the amount of glucose in the culture medium. Therefore, the concentration of glucose can be measured by counting the cell number after culturing the bacteria. With Davis medium depleted of sodium citrate, we used this method to detect a subnanogram level of glucose, although with the accuracy of order of magnitude estimation. In the microgram per milliliter range of glucose, the cell number can be measured optically, with proliferation being proportional to the input glucose. The high selectivity for glucose of this method is based on the preferential usage of glucose as a carbon and energy source by the bacteria adopted. The feasibility of applying this method to other substances is discussed.

Bacteriological Techniques↗
PubMed1986

Detection of differences in higher order structure between highly homologous single-stranded DNAs by low-temperature denaturant gradient gel electrophoresis.

Denaturant gradient gel electrophoresis performed at low temperature was shown to be able to detect the mobility change corresponding to the denaturation of the secondary structure of single-stranded(ss)DNA. Mobility transitions observed were determined to correspond to the melting of local structures, since both denaturants and temperature were verified to have similar effects on the mobility transition of single-stranded DNAs as on that of double-stranded DNAs. In this study it was found that point mutations can effectively change the secondary structures of ssDNA and RNA, and that the method adopted here is very sensitive to such alterations. The validity of the method was supported by a computer analysis of the secondary structure of DNA.

DNA, Single-Stranded↗
PubMed1985

Type II restriction endonucleases cleave single-stranded DNAs in general.

Restriction endonucleases (13 out of 18 species used for the test) were certified to cleave single-stranded(ss)DNA. Such enzymes as AvaII, HaeII, DdeI, AluI, Sau3AI, AccII,TthHB8I and HapII were newly reported to cleave ssDNA. A model to account for the cleavage of ssDNA by restriction enzymes was proposed with supportive data. The essential part of the model was that restriction enzymes preferentially cleave transiently formed secondary structures (called canonical structures) in ssDNA composed of two recognition sequences with two fold rotational symmetry. This means that a restriction enzyme can cleave ssDNAs in general so far as the DNAs have the sequences of restriction sites for the enzyme, and that the rate of cleavage depends on the stabilities of canonical structures.

Base Sequence↗
PubMed1984

Strand dissociation and cooperative melting of double-stranded DNAs detected by denaturant gradient gel electrophoresis.

Precise analysis using the denaturant gradient gel electrophoresis which was devised by Fischer and Lerman (Cell 16, 191-200, 1979), was found to present specific patterns of fine structures for double stranded DNA fragments. These seem to be a mobility change of DNA fragments caused by specific denaturation processes. The effect of denaturants on DNA melting was ascertained to be similar to the effect of temperature. The observed patterns, in comparison with the melting processes of DNAs theoretically obtained, were closely related to DNA meltings and strand dissociations. A number of electrophoretic mobility transitions showed the retardation correspondent to each of the cooperative meltings. Strand dissociations occurred under the conditions theoretically predicted. The degree of retardation in electrophoresis for DNA fragments seemed to correspond to the size of melted regions. Methods presented here were proved to have advantages over the conventional ones for the study of DNA stability maps.

Computers↗
PubMed1980

Fine structure in the thermal denaturation of DNA: high temperature-resolution spectrophotometric studies.

Fine structures which appear in the optical melting profile of DNA are examined from both the experimental and theoretical aspects. After a brief historical survey of the DNA melting experiments during the pre-fine-structure era in Section II, the high temperature-resolution experimental techniques which are essential to the investigation of fine structure are described in Section III. Then, the current status of the high-resolution study is reviewed first by a phenomenological description of the melting profile (Section IV) and then of the refolding profile (Section V), where a general idea about the cooperatively melting region and several factors affecting it is given. Sections VI and VII are devoted to the review of current theoretical works. Several well-established theoretical frameworks which correlate the base sequence with the melting phenomena are examined in terms of their rigorousness and usefulness. The molecular thermodynamic parameters concerning the DNA melting which have been evaluated by several research groups are compared and discussed. Finally, in Section VIII, current ideas on the correlation between the fine structure and genetic functions and genetic maps are reviewed. Some future problems relating to the fine structure are also discussed.

Animals↗
PubMed1979

Stability mapping along the DNA double strand and its relation to the genetic map.

The distribution of the stability of the double helical structure along the whole DNA of fdphage and its restriction fragments is calculated. In this calculation, Poland's method, which has been established as a rigorous algorithm for taking the base sequence explicitly into consideration, is used. The molecular thermodynamic parameters in the calculation have been determined so as to best reproduce the melting profile of the DNA and its fragments. The results, which are presented as melting maps, show fairly good agreement with those experimentally obtained by the present authors earlier. A close correlation with genes in the genetic map is apparent for some cooperatively melting regions observed in the stability map.

Chemical Phenomena↗
PubMed1978

"Thermal stability" maps for several double-stranded DNA fragments of known sequence.

The origin of cooperatively melting regions in DNA, which appear as fine structures in the optical melting profile, has been examined for DNA fragments of known base sequences from bacteriophages phiX174 and fd. Thermal stability maps, which indicate the states of base pairs along these DNA strands, were constructed within the established theoretical framework using the parameters which best reproduce the melting profiles obtained by high temperature resolution experiments. By comparing these stability maps with genetic maps, it was found that several cooperatively melting regions which span several hundred bases have some correlation with the gene locations.

Base Sequence↗
PubMed1977

Kinetics of the polymerization reaction of tobacco mosaic virus protein: transient-saturation type polymerization reaction.

The kinetics of the endothermic polymerization reaction of tobacco mosaic virus protein in the mild acid region was studied by means of temperature-jump (rising time of 6 sec)-turbidimetry, electron microscopy, and computer simulation. The time course profile of the turbidity increase changed from a normal one to an anomalous one as the size of the temperature-jump was made greater. The anomalous type polymerization profile, which we named the "transient-saturation" type, could be characterized by a rapid increase of turbidity and its transient saturation, and a slow increase to the final level. At a higher concentration of the protein, this transient-saturation effect was more marked, whereas the slow turbidity in the second phase occurred with a higher rate. This transient-saturation type polymerization profile was observed also in a pH-induced polymerization reaction. It was not observed in the case of the N-bromosuccinimide modified tobacco mosaic virus protein under a similar environmental change. By an electron microscopic study and computer simulation, it was revealed that in the first phase, a large number of short polymers were formed, and the concentration of the polymerizing units was rapidly reduced to the equilibrium value, and the polymerization reaction stopped transiently. In the second phase, polymer-polymer associations took place slowly and longer polymers were formed. The revlevance of the present study to the polymerization reaction of actin, myosin, and to a transient-overshoot type polymerization are discussed.

Bromosuccinimide↗