Biomedical subjects

Toshihiko Ashikari

Publications and source records attributed to Toshihiko Ashikari.

10 recordsLinked to original sources

PubMed2006

Screening for candidate genes involved in tolerance to organic solvents in yeast.

Saccharomyces cerevisiae mutant strain, KK-211, isolated from serial culture in medium containing isooctane showed an extremely higher tolerance to the hydrophobic organic-solvents, which are toxic to yeast cells compared to the wild-type parent strain, DY-1. To detect genes that are related to this tolerance, a DNA microarray analysis was performed using mRNAs isolated from strains DY-1 and KK-211. Fourteen genes were identified as being related to the tolerance. The expression of 12 genes including ICT1, YNL190W, and PRY3, was induced while the expression of two genes including PHO84 was repressed in strain KK-211. Two genes, ICT1 and YNL190W showed the same profile in the DNA microarray analysis and a differential display-polymerase chain reaction analysis. But, there is no detectable difference in the expression profile of KK-211 cells cultured with or without isooctane. The results suggest that change in expression levels of multiple genes that confer the modification function of the cell surface, not by a single gene, might be required for yeast cell tolerance to organic solvents.

Culture Media↗

PubMed2006

Pure and mixed genetic lines of Saccharomyces bayanus and Saccharomyces pastorianus and their contribution to the lager brewing strain genome.

The yeast species Saccharomyces bayanus and Saccharomyces pastorianus are of industrial importance since they are involved in the production process of common beverages such as wine and lager beer; however, they contain strains whose variability has been neither fully investigated nor exploited in genetic improvement programs. We evaluated this variability by using PCR-restriction fragment length polymorphism analysis of 48 genes and partial sequences of 16. Within these two species, we identified "pure" strains containing a single type of genome and "hybrid" strains that contained portions of the genomes from the "pure" lines, as well as alleles termed "Lager" that represent a third genome commonly associated with lager brewing strains. The two pure lines represent S. uvarum and S. bayanus, the latter a novel group of strains that may be of use in strain improvement programs. Hybrid lines identified include (i) S. cerevisiae/S. bayanus/Lager, (ii) S. bayanus/S. uvarum/Lager, and (iii) S. cerevisiae/S. bayanus/S. uvarum/Lager. The genome of the lager strains may have resulted from chromosomal loss, replacement, or rearrangement within the hybrid genetic lines. This study identifies brewing strains that could be used as novel genetic sources in strain improvement programs and provides data that can be used to generate a model of how naturally occurring and industrial hybrid strains may have evolved.

Beer↗

PubMed2005

Molecular evidence that the rate-limiting step for the biosynthesis of arachidonic acid in Mortierella alpina is at the level of an elongase.

The oil-producing fungus Mortierella alpina 1S-4 is an industrial strain for arachidonic acid (AA) production. To determine its physiological properties and to clarify the biosynthetic pathways for PUFA, heterologous and homologous gene expression systems were established in this fungus. The first trial was performed with an enhanced green fluorescent protein gene to assess the transformation efficiency for heterologous gene expression. As a result, strong fluorescence was observed in the spores of the obtained transformant, suggesting that the foreign gene was inherited by the spores. The next trial was performed with a homologous PUFA elongase (GLELOp) gene, this enzyme having been reported to catalyze the elongation of GLA (18:3n-6) to dihomo-gamma-linolenic acid (20:3n-6), and to be the rate-limiting step of AA production. The FA composition of the transformant was different from that of the host strain: The GLA content was decreased whereas that of AA was increased. These data support the hypothesis that the GLELOp enzyme plays an important role in PUFA synthesis, and may indicate how to control PUFA biosynthesis.

Acetyltransferases↗

PubMed2005

Improvement of the fatty acid composition of an oil-producing filamentous fungus, Mortierella alpina 1S-4, through RNA interference with delta12-desaturase gene expression.

An oleaginous fungus, Mortierella alpina 1S-4, is used commercially for arachidonic acid production. Delta12-Desaturase, which desaturates oleic acid (18:1n-9) to linoleic acid (18:2n-6), is a key enzyme in the arachidonic acid biosynthetic pathway. To determine if RNA interference (RNAi) by double-stranded RNA occurs in M. alpina 1S-4, we silenced the Delta12-desaturase gene. The silenced strains accumulate 18:2n-9, 20:2n-9, and Mead acid (20:3n-9), which are not detected in either the control strain or wild type strain 1S-4. The fatty acid composition of stable transformants was similar to that of Delta12-desaturation-defective mutants previously identified. Thus, RNAi occurs in M. alpina and could be used to alter the types and relative amounts of fatty acids produced by commercial strains of this fungus without mutagenesis or other permanent changes in the genetic background of the producing strains.

Arachidonic Acid↗

PubMed2004

Establishment of an overall transformation system for an oil-producing filamentous fungus, Mortierella alpina 1S-4.

Oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. To determine its physiological properties and to clarify the biosynthetic pathways for polyunsaturated fatty acids, a transformation system for this fungus was established using a derivative of it, i.e., a ura5- mutant lacking orotate phosphoribosyl transferase (OPRTase, EC.2.4.2.10) activity. Transformation with a vector containing the homologous ura5 gene as a marker was successfully performed using microprojectile bombardment, other methods frequently used for transformation, such as the protoplasting, lithium acetate, or electroporation methods, not giving satisfactory results. As a result, two types of transformants were obtained: a few stable transformants overexpressing the ura5 gene, and many unstable transformants showing OPRTase activity comparable to that of the wild-type strain. The results of quantitative PCR indicated that the stable transformants could retain the ura5 genes originating from the transformation vector regardless of the culture conditions. On the other hand, unstable transformants easily lost the marker gene under uracil-containing conditions, as expected. In this paper, we report that an overall transformation system for this fungus was successfully established, and propose how to select useful transformants as experimental and industrial strains.

Acetates↗

PubMed2004

Cloning and sequencing of the ura3 and ura5 genes, and isolation and characterization of uracil auxotrophs of the fungus Mortierella alpina 1S-4.

The oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. In order to prepare host strains for a transformation system for this fungus, six uracil auxotrophs were obtained by means of random mutation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). When the activities of orotate phosphoribosyl transferase (OPRTase, EC 2.4.2.10) and orotidine-5'-phosphate decarboxylase (OMPdecase, EC 4.1.1.23) were examined in the mutants and wild strain, OPRTase activity was found to be completely absent in all mutants, on the other hand, OMPdecase activity was intact. The genomic DNA and cDNA of the ura5 gene encoding OPRTase and the ura3 gene encoding OMPdecase were cloned and sequenced. The Ura5p deduced amino acid sequence of this fungus showed highest similarity to that of Vibrio cholerae classed among prokaryote. Furthermore, the mutational points in the ura5 genes of two selected mutants were identified; a base-replacement and a base-insertion.

Amino Acid Sequence↗

PubMed2003

S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex.

The checkpoint regulatory mechanism has an important role in maintaining the integrity of the genome. This is particularly important in S phase of the cell cycle, when genomic DNA is most susceptible to various environmental hazards. When chemical agents damage DNA, activation of checkpoint signalling pathways results in a temporary cessation of DNA replication. A replication-pausing complex is believed to be created at the arrested forks to activate further checkpoint cascades, leading to repair of the damaged DNA. Thus, checkpoint factors are thought to act not only to arrest replication but also to maintain a stable replication complex at replication forks. However, the molecular mechanism coupling checkpoint regulation and replication arrest is unknown. Here we demonstrate that the checkpoint regulatory proteins Tof1 and Mrc1 interact directly with the DNA replication machinery in Saccharomyces cerevisiae. When hydroxyurea blocks chromosomal replication, this assembly forms a stable pausing structure that serves to anchor subsequent DNA repair events.

Bromodeoxyuridine↗

PubMed2002

Genome-wide expression analysis of genes affected by amino acid sensor Ssy1p in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Ssy1p is a membrane protein which senses extracellular amino acids and controls the expression of certain amino acid permease genes. Analysis by DNA micro-array newly identified DIP5 and MUP1 as the positive targets and CAN1, PUT4 and GAP1 as the negative targets under Ssy1p control. Interestingly, the effect of ssy1 deletion was not restricted to amino acid permease genes: the expression of nitrogen catabolite repression (NCR)-sensitive genes and methionine-biosynthesizing genes ( MET genes) was derepressed by the deletion of SSY1. Constitutive overexpression of the genes for glutamine permease ( GNP1) or methionine permease ( MUP1) enhanced the assimilation of glutamine or methionine in the ssy1Delta strain but could not fully suppress the derepression of the NCR-sensitive genes or MET genes. This result suggests that Ssy1p regulates not only the transcription of amino acid permease genes, but also transcription of many other nitrogen-metabolizing genes.

Amino Acid Transport Systems↗

PubMed2002

Deletion and insertion of a 192-residue peptide in the active-site domain of glycosyl hydrolase family-2 beta-galactosidases.

The monomeric multimetal-binding beta-galactosidase of Saccharopolyspora rectivirgula (srbg), a glycosyl hydrolase family-2 enzyme, has a unique sequence consisting of 192 amino acid residues with no similarity to known proteins. This 192-residue sequence (termed the "iota [iota] sequence") appears to be inserted into a sequence homologous to the active-site domain of the Escherichia coli lacZ enzyme (lacZbg). To assess the effects of the t sequence at specific sites of beta-galactosidase on the catalytic functioning and molecular properties of beta-galactosidase, deletion or insertion mutants of beta-galactosidases were constructed, expressed in LacZ- E. coli strains, and characterized: srbgdelta in which the iota sequence was deleted from srbg, and lacZbgI, in which the 192-residue iota sequence was inserted into the corresponding position (between Asp591 and Phe592) in the active-site domain of lacZbg. srbgdelta was a catalytically inactive, dimeric protein which retained multimetal-binding characteristics, suggesting that the iota sequence is very important for maintaining the structure necessary for the catalytic functioning and the monomeric structure of srbg but is not responsible for the unique metal ion requirements of srbg. On the other hand, lacZbgI existed as a mixture of a monomer, a tetramer, and higher multimers. The monomeric species was inactive, whereas the tetramer and other multimers were catalytically active (V(max )K(m) value, 25% of that of lacZbg) and highly specific for beta-D-galactoside. The tetrameric lacZbgI was activated by Mg2+ and Mn2+ with lowered metal affinities, and the stoichiometry of metal binding was unchanged from that of lacZbg. These results, along with the published stereo structure of lacZbg, suggest that, in lacZbgI, the inserted 192-residue iota peptide could fold independently of the lacZbg domains into a "sub-domain," lying distant from the active site and subunit interfaces.

Journal Article↗

PubMed2002

Influence of moderate drinking on purine and carbohydrate metabolism.

BACKGROUND: We examined the influences of a moderate intake level of three types of alcoholic beverages--beer, whisky, and Shochu (Japanese distilled liquor)--on purine and carbohydrate metabolism and excretion in healthy male volunteers, concerning (1) the extent of contribution of purine bodies contained in beer to uric acid metabolism and (2) a comparison between two types of distilled spirits with (whisky) and without (Shochu) aging in oak wood barrel storage. METHODS: Three sets of studies were conducted in which 10 to 13 healthy adult men were instructed to drink three types of alcoholic beverages at a slightly higher level (0.8 ml of ethanol equivalent/kg body weight) than moderate drinking (approximately 30.4 ml or less for men). A low purine beer was test-manufactured by treating nucleosides that were contained in wort and remained in beer with purine nucleoside phosphorylase derived from Ochrobacterium anthropi, thereby converting them into corresponding purine bases that were easily assimilated by beer yeast. RESULTS: Although beer intake enhanced the level of serum uric acid by 13.6%, blood glucose by 26.7%, and insulin level by 5.1-fold, drinking a moderate level of distilled liquor (whisky, Shochu) did not increase the serum uric acid level or the other two parameters. The serum uric acid level observed after drinking beer with a purine body concentration reduced by 28% (68% in nucleosides and purine bases) was almost identical to the level observed after drinking regular beer. Whisky has been found to have a property that decreases the serum uric acid level. Excretion of uric acid from blood is increased by 27% after drinking whisky. CONCLUSIONS: Moderate drinking of distilled liquors did not enhance serum uric acid level, blood glucose, or insulin level in healthy male subjects. Increased serum uric acid after beer intake could not be explained mostly with their purine body congeners. Whisky showed the eliminative property in serum uric acid through excretion of it from blood to urine. At a moderate drinking level, beer and whisky have different effects on purine metabolism or excretion.

Adult↗