Biomedical subjects

Satoshi Harashima

Publications and source records attributed to Satoshi Harashima.

At least 19 recordsLinked to original sources

PubMed2006

Chromosome XII context is important for rDNA function in yeast.

The rDNA cluster in Saccharomyces cerevisiae is located 450 kb from the left end and 610 kb from the right end of chromosome XII and consists of approximately 150 tandemly repeated copies of a 9.1 kb rDNA unit. To explore the biological significance of this specific chromosomal context, chromosome XII was split at both sides of the rDNA cluster and strains harboring deleted variants of chromosome XII consisting of 450 kb, 1500 kb (rDNA cluster only) and 610 kb were created. In the strain harboring the 1500 kb variant of chromosome XII consisting solely of rDNA, the size of the rDNA cluster was found to decrease as a result of a decrease in rDNA copy number. The frequency of silencing of URA3 inserted within the rDNA locus was found to be greater than in a wild-type strain. The localization and morphology of the nucleolus was also affected such that a single and occasionally (6-12% frequency) two foci for Nop1p and a rounded nucleolus were observed, whereas a typical crescent-shaped nucleolar structure was seen in the wild-type strain. Notably, strains harboring the 450 kb chromosome XII variant and/or the 1500 kb variant consisting solely of rDNA had shorter life spans than wild type and also accumulated extrachromosomal rDNA circles. These observations suggest that the context of chromosome XII plays an important role in maintaining a constant rDNA copy number and in physiological processes related to rDNA function in S.cerevisiae.

Cell Nucleolus↗

PubMed2006

Chromosome-shuffling technique for selected chromosomal segments in Saccharomyces cerevisiae.

We describe a novel chromosome engineering technique for shuffling selected regions of chromosomes from two strains in Saccharomyces cerevisiae: The technique starts with the construction of MATa and MATalpha strains in which a particular chromosome is split at exactly the same site in both strains such that the split chromosomes generated are marked with different markers. The two strains are then crossed, and the resultant diploid is cultivated in nutrient medium to induce loss of the split chromosome originating from either of the strains. We predicted that some of these clones that are hemizygous for the split chromosome would spontaneously restore a homozygous configuration of the split chromosome during cultivation. We verified this prediction by tetrad analysis and quantitative Southern hybridization analysis, indicating that it is possible to create diploid hybrids in which a selected region of a chromosome from one strain is replaced by the corresponding chromosomal region from another strain. We also found that some chromosomal segments maintain a hemizygous state. This novel technique, which we call 'chromosome shuffling', could provide a new tool to analyze phenotypic alterations caused by the replacement or hemizygosity of a selected chromosomal region in not only laboratory but also industrial strains of S. cerevisiae.

Blotting, Southern↗

PubMed2006

A polymerase chain reaction-mediated yeast artificial chromosome-splitting technology for generating targeted yeast artificial chromosomes subclones.

The yeast artificial chromosomes (YAC) system makes it possible not only to clone large DNA fragments but also to simplify the physical mapping and functional analysis of chromosomes and genomes through YAC manipulation. To manipulate large DNA fragments cloned into YACs, YAC fragmentation has been developed and also a new method called the YAC-splitting method was developed recently. Although the YAC fragmentation method can be used to delete DNA from one side of a YAC, the YAC-splitting method generates two smaller YACs, both of which are transmitted to daughter cells during mitotic growth. This chapter describes the YAC-splitting method improved by incorporating polymerase chain reaction-mediated chromosome splitting (PCS) technique and by adding yeast autonomously replicating sequence (ARS) to the system. The PCS method combines a streamlined procedure (two-step PCR and one transformation per splitting event) with the Cre/loxP system for marker rescue. The improved YAC-splitting method can convert a targeted region of a eukaryotic chromosome within a YAC into a new replicating YAC.

Chromosomes, Artificial, Yeast↗

PubMed2006

Transformation of yeast using bioactive beads with surface-immobilized yeast artificial chromosomes.

Yeast artificial chromosomes (YACs) are useful cloning vectors with the capacity to carry large DNA inserts. The largest barrier using such large DNA molecules in transformation experiments has been their physical instability in a solution. We developed a new method for transforming yeast with chromosome-sized DNA. The method uses bioactive beads composed of calcium alginate to immobilize yeast chromosomal DNAs. Chromosomal DNA immobilized on bioactive beads is physically stable when compared with naked chromosomal DNAs. The bead-mediated transformation performed well, not only with respect to the transformation frequency, but also in successful transformation using split chromosomal DNA that exceeded 450 kb in size. In this chapter we introduce a new method for transforming yeast using bioactive beads. In conjunction with genomic YAC libraries and the yeast chromosome-splitting method, this technique will pave the way to stable and effective transfer of YACs into yeast cells.

Alginates↗

PubMed2005

A versatile and general splitting technology for generating targeted YAC subclones.

Yeast artificial chromosomes (YAC) splitting technology was developed as a means to subclone any desired region of eukaryotic chromosomes from one YAC into new YACs. In the present study, the conventional YAC splitting technology was improved by incorporating PCR-mediated chromosome splitting technique and by adding autonomously replicating sequence (ARS) to the system. To demonstrate the performance of the improved method, a 60-kb region from within a 590-kb YAC (clone CIC9e2 from Arabidopsis thaliana chromosome 5) that could not be subcloned using the original method was split to convert into a replicating YAC. Two template plasmids, pSK-KCA and pSKCLY, were used to generate two splitting fragments by PCR. Two splitting fragments consisted of telomeric (C(4)A(2))(6) repeats, 400-bp target region, CEN4, H4ARS and Km(r) (selective marker for plant transformants), or CgLEU2. These splitting fragments were introduced into Saccharomyces cerevisiae harboring the 100-kb split YAC generated by splitting of the 590-kb YAC and containing the 60-kb region. Among 12 Leu(+) transformants, four exhibited the expected karyotype in which two newly split 40- and 60-kb chromosomes were generated. These results demonstrate that the improved method can convert a targeted region of a eukaryotic chromosome within a YAC into a replicating YAC.

Arabidopsis↗

PubMed2005

Plc1p, Arg82p, and Kcs1p, enzymes involved in inositol pyrophosphate synthesis, are essential for phosphate regulation and polyphosphate accumulation in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the phosphate signal transduction PHO pathway is involved in regulating several phosphate-responsive genes such as PHO5, which encodes repressible acid phosphatase. In this pathway, a cyclin-dependent kinase inhibitor (Pho81p) regulates the kinase activity of the cyclin-cyclin-dependent kinase complex Pho80p-Pho85p, which phosphorylates the transcription factor Pho4p in response to intracellular phosphate levels. However, how cells sense phosphate availability and transduce the phosphate signal to Pho81p remains unknown. To identify additional components of the PHO pathway, we have screened a collection of yeast deletion strains. We found that disruptants of PLC1, ARG82, and KCS1, which are involved in the synthesis of inositol polyphosphate, and ADK1, which encodes adenylate kinase, constitutively express PHO5. Each of these factors functions upstream of Pho81p and negatively regulates the PHO pathway independently of intracellular orthophosphate levels. Overexpression of KCS1, but not of the other genes, suppressed PHO5 expression in the wild-type strain under low phosphate conditions. These results raise the possibility that diphosphoinositol tetrakisphosphate and/or bisdiphosphoinositol triphosphate may be essential for regulation of the PHO pathway. Furthermore, the Deltaplc1, Deltaarg82, and Deltakcs1 deletion strains, but not the Deltaipk1 deletion strain, had significantly reduced intracellular polyphosphate levels, suggesting that enzymes involved in inositol pyrophosphate synthesis are also required for polyphosphate accumulation.

Adenosine Triphosphate↗

PubMed2005

The SIT4 gene, which encodes protein phosphatase 2A, is required for telomere function in Saccharomyces cerevisiae.

Life span and number of cell divisions in eukaryotes are limited. The accumulation of stress-associated damage due to ageing may cause irreversible cell cycle arrest, so-called "cellular senescence". Although many genes have been implicated in determining life span, regulatory systems that counteract age-related stress have not yet been clarified. We examined senescence during a stress of Saccharomyces cerevisiae strains carrying disruptions in protein phosphatase (PPase)-encoding genes in order to identify the system counteracting senescence. Among these strains, short telomeres were found in the sit4 disruptant that lacks one form of protein phosphatase 2A (PP2A). Silencing ability in the subtelomeric region was impaired and hyperphosphorylation of Sir3 was also observed in this mutant. The sit4 mutant was found to have altered nucleoli and a life span as short as an sgs1 mutant. These observations suggest that the PP2A pathway regulates life span in yeast.

Cell Cycle↗

PubMed2005

A yeast artificial chromosome-splitting vector designed for precise manipulation of specific plant chromosome region.

A yeast artificial chromosome (YAC) splitting vector, pKI01, was constructed for manipulating plant chromosome fragments cloned as YACs in order to transfer specific regions of the fragments into plant cells. Vector pKI01 consists of Km(r) and ADE2 genes (selective markers for plant and yeast transformants, respectively), inverted telomeric repeats Tr and CEN4. To demonstrate the utility of pKI01, YAC CIC9e2 harboring a 590-kb fragment from Arabidopsis thaliana chromosome 5 was split into specific fragments. A 1-kb target region positioned 100 kb from the right end of the 590 kb fragment was cloned into pKI01. The resultant plasmid, pKY03, was introduced into Saccharomyces cerevisiae harboring YAC CIC9e2. The Ade+ transformants were found to contain two new YACs of 490 and 100 kb, and to lack the original 590 kb YAC, consistent with the expected splitting event. To release the desired middle region of YAC CIC9e2, two additional splitting vectors were constructed, pKY11 and pKY14. By conducting two rounds of splitting, i.e., the first round 100 kb from the right end of YAC CIC9e2 with pKY11 to generate 490 and 100 kb YACs and a second round 50 kb from the right end of the new 490 kb YAC to generate 440 and 50 kb YACs, the middle 50 kb region of a plant chromosome fragment harboring Km(r) was successfully released as a split YAC. These results indicate that YAC splitting vectors as constructed in this study are useful for generating any desired plant chromosome fragment as a YAC for eventual re-introduction into plant cells.

Arabidopsis↗

PubMed2005

PCR-mediated repeated chromosome splitting in Saccharomyces cerevisiae.

Chromosome engineering is playing an increasingly important role in the functional analysis of genomes. A simple and efficient technology for manipulating large chromosomal segments is key to advancing these analyses. Here we describe a simple but innovative method to split chromosomes in Saccharomyces cerevisiae, which we call PCR-mediated chromosome splitting (PCS). The PCS method combines a streamlined procedure (two-step PCR and one transformation per splitting event) with the CreAoxP system for marker rescue. Using this novel method, chromosomes I (230 kb) and XV (1091 kb) of a haploid cell were split collectively into 10 minichromosomes ranging in size from 29-631 kb with high efficiency (routinely 80%) that were occasionally lost during mitotic growth in various combinations. These observations indicate that the PCS method provides an efficient tool to engineer the yeast genome and may offer a possible approach to identify minimal genome constitutions as a function of culture conditions through further splitting, followed by combinatorial loss of minichromosomes.

Blotting, Southern↗

PubMed2004

Obtaining transgenic plants using the bio-active beads method.

Several methods of transformation are currently available for delivering exogenous DNA into animal and plant cells. In this study, a novel and efficient transformation system for DNA delivery/expression with a capacity to transport DNA of high molecular weight was developed. This system can overcome the shortcomings of traditional transformation methods such as Agrobacterium-mediated transformation, particle bombardment, and the electroporation method. The method developed in this study uses calcium alginate micro beads to immobilize DNA molecules in combination with polyethylene glycol treatment. In addition, it is simple and low-cost, and requires limited equipment. Using this method, we have successfully transformed tobacco plants, screening by kanamycin resistance. The transformed genes in the transformants were confirmed by PCR and Southern hybridization.

Alginates↗

PubMed2004

Intracellular phosphate serves as a signal for the regulation of the PHO pathway in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the phosphate signal transduction pathway (PHO pathway) is known to regulate the expression of several phosphate-responsive genes, such as PHO5 and PHO84. However, the fundamental issue of whether cells sense intracellular or extracellular phosphate remains unresolved. To address this issue, we have directly measured intracellular phosphate concentrations by (31)P NMR spectroscopy. We find that PHO5 expression is strongly correlated with the levels of both intracellular orthophosphate and intracellular polyphosphate and that the signaling defect in the Deltapho84 strain is likely to result from insufficient intracellular phosphate caused by a defect in phosphate uptake. Furthermore, the Deltaphm1Deltaphm2, Deltaphm3, and Deltaphm4 strains, which lack intracellular polyphosphate, have higher intracellular orthophosphate levels and lower expression of PHO5 than the wild-type strain. By contrast, the Deltaphm5 strain, which has lower intracellular orthophosphate and higher polyphosphate levels than the wild-type strain, shows repressed expression of PHO5, similar to the wild-type strain. These observations suggest that PHO5 expression is under the regulation of intracellular orthophosphate, although orthophosphate is not the sole signaling molecule. Moreover, the disruption of PHM3, PHM4, or of both PHM1 and PHM2 in the Deltapho84 strain suppresses, although not completely, the PHO5 constitutive phenotype by increasing intracellular orthophosphate, suggesting that Pho84p affects phosphate signaling largely by functioning as a transporter.

Biological Transport↗

PubMed2003

Transcriptional regulation of phosphate-responsive genes in low-affinity phosphate-transporter-defective mutants in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, two systems have been shown to be involved in the active transport of inorganic phosphate (P(i)) across the plasma membrane, the high-affinity system and the low-affinity system. The high-affinity system consists of Pho84p and Pho89p. The low-affinity system has recently been shown to be composed of Pho87p, Pho90p, and Pho91p. In this study, we found that the Deltapho87Deltapho90Deltapho91 strain which shows repressed PHO5 expression under high-P(i) condition has, unlike the wild-type strain, increased levels of PHO5 expression at an intermediate P(i) concentration of 0.5mM, whereas it is not defective in terms of P(i) uptake under the same conditions. Moreover, we observed that the transcription levels of PHO84 and PHO89 are also increased in low-affinity P(i)-transporter-defective mutants, indicating that the inactivation of low-affinity P(i) transporters leads to the activation of the PHO pathway. In contrast to that of PHO5, PHO84, and PHO89, the transcription of PHO87, PHO90, and PHO91 genes is independent of P(i) concentration and Pho4p activity, and the increased expression level of these transporters does not occur when other transporters including PHO84 are inactivated. The fact that low-affinity P(i)-transporter-defective mutants exhibit a derepression of P(i)-responsive genes suggests that low-affinity transporters play a role not only in P(i) transport but also in the regulation of the P(i) signal transduction pathway.

Biological Transport↗

PubMed2003

Creating a Saccharomyces cerevisiae haploid strain having 21 chromosomes.

Chromosome engineering techniques that can manipulate a large segment of chromosomal DNA are useful not only for studying the organization of eukaryotic genomes but also for the improvement of industrially important strains. Toward the development of techniques that can efficiently manipulate a large segment of chromosome, we have previously reported a one-step chromosome splitting technique in a haploid Saccharomyces cerevisiae cell, with which we could successfully split yeast chromosome 11, XIII, or XI into two halves to create a haploid strain having 17 chromosomes. We have now constructed chromosome splitting vectors bearing ADE2, HIS3, LEU2, or TRP1 marker, and by using these vectors, we could successively split yeast chromosomes to create a novel yeast haploid strain having up to 21 chromosomes. The specific growth rates of yeast strains carrying more than 16 chromosomes up to 21 did not differ significantly, suggesting that yeast cells can harbor more chromosomes than they do in their natural state, that is, 16 chromosomes, without serious effects on their growth.

Journal Article↗

PubMed2003

Repeated chromosome splitting targeted to delta sequences in Saccharomyces cerevisiae.

We have previously developed a chromosome-splitting technique based on homologous recombination in Saccharomyces cerevisiae. To facilitate chromosome splitting at multiple sites, we focused on the delta sequences that are distributed in more than 200 copies throughout the yeast genome. We constructed a new chromosome-splitting vector harboring the YFLWdelta4 sequence and the hisG-URA3-hisG cassette, and transformed yeast cells with this vector. The karyotype analysis of transformants showed that chromosomes XIV, III, and IV, or other chromosomes are split. After the excision of the URA3 gene, the transformant with split chromosome IV was subsequently transformed with the same vector. Karyotype analysis revealed that further splitting occurred at chromosome X, the split chromosome IV, or other chromosomes. These results indicate that delta sequences are efficient target sites for repeated chromosome splitting at multiple sites with a single vector.

Journal Article↗

PubMed2003

Transformation of yeast using calcium alginate microbeads with surface-immobilized chromosomal DNA.

Yeast artificial chromosomes (YACs) are useful cloning vectors that have the capacity to carry large DNA inserts. The largest barrier to using such large DNA molecules in transformation experiments has been their physical instability in solution. We developed a new method of transforming yeast using chromosome-sized DNA. The method uses calcium alginate microbeads to immobilize high-density yeast chromosomal DNA. Chromosomal DNA immobilized on microbeads is physically stabilized compared with naked chromosomal DNA. The microbead-mediated transformation performed well, not only with respect to the transformation frequency with large DNA molecules (> 100 kb) but also in successful transformation using split chromosome DNA that exceeded 450 kb.

Adsorption↗

PubMed2002

Chemo-enzymatic synthesis of the glycosylated alpha-mating factor of Saccharomyces cerevisiae and analysis of its biological activity.

The effect of glycosylation on a bioactive peptide was studied using yeast Saccharomyces cerevisiae alpha-mating factor, which is composed of 13 amino acids. In this study, we prepared glycosylated alpha-mating factor by chemo-enzymatic synthesis. At first, N-acetylglucosaminyl alpha-mating factor (Trp-His-Trp-Leu-Gln(GlcNAc)-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) was chemically synthesized by the solid-phase method. Then, using the transglycosylation activity of Mucor hiemalis endo-beta-N-acetylglucosaminidase, we synthesized glycosylated alpha-mating factor with a glutamine-linked sialo complex type oligosaccharide. The biological activity of alpha-mating factor derivatives was examined by means of a growth arrest assay using secreted-protease-defective a cells of S. cerevisiae. The results showed that the bioactivity of glycosylated alpha-mating factor was lower than that of native alpha-mating factor. However, when sialic acid was removed from the complex type sugar chain of glycosylated alpha-mating factor, its bioactivity was recovered. Glycosylated alpha-mating factor exhibited higher resistance against proteolysis than native alpha-mating factor. It was found that the bioactivity of N-acetylglucosaminyl alpha-mating factor was higher than that of alpha-mating factor. Circular dichroism studies indicated that a slight change in the structure of alpha-mating factor may influence its activity.

Acetylglucosamine↗

PubMed2002

Mga2p is a putative sensor for low temperature and oxygen to induce OLE1 transcription in Saccharomyces cerevisiae.

Various low-temperature-inducible genes such as fatty acid desaturase genes are essential for all living organisms to acclimate to low temperature. However, a low-temperature signal transduction pathway has not been identified in eukaryotes. In yeast Saccharomyces cerevisiae, the Delta9 fatty acid desaturase gene OLE1 is activated by ubiquitin/proteasome-dependent processing of two homologous endoplasmic reticulum membrane proteins, Spt23p and Mga2p. We found that OLE1 transcription was transiently activated with resultant increases in the degree of unsaturation of total fatty acids when culture temperature was downshifted from 30 degrees C to 10 degrees C. This activation was greatly depressed in Deltamga2 cells. Although Mga2p is essential for hypoxic activation of OLE1 transcription, and its hypoxic functions are repressed by unsaturated fatty acids (UFAs), low-temperature activation of the OLE1 gene was not repressed by UFAs. These observations suggest that low-temperature and hypoxic signal transduction pathways share some components, and Mga2p is the first identified eukaryotic sensor for low temperature and oxygen.

Cell Hypoxia↗

PubMed2002

A series of double disruptants for protein phosphatase genes in Saccharomyces cerevisiae and their phenotypic analysis.

Thirty-two protein phosphatase (PPase) genes were identified in Saccharomyces cerevisiae based on the nucleotide sequences of the entire genome. In an effort to understand the role of PPases and their functional redundancy in the cellular physiology of one of the reference eukaryotic organisms, a series of single and double PPase gene disruptants were constructed in the W303 strain background. Two single disruptants for the CDC14 and GLC7 genes were lethal. Double disruptants for 30 non-essential PPase genes were constructed in all possible 435 combinations. No double disruptant showed synthetic lethality. Several phenotypes of the viable 30 single and 435 double disruptants were examined; temperature-sensitive growth, utilization of carbon sources and sensitivity to cations and drugs. Four double disruptants exhibited synthetic phenotypes in addition to eight single ones: the pph21 pph22 double disruptant showed slow growth on complete medium, as did the sit4 and yvh1 single ones. In addition to the ptc1, ynr022c and ycr079w single disruptants, the ppz1 ppz2 double disruptant showed temperature-sensitive slow growth. The msg5 ptp2 double disruptant, like the ynr022c single one, did not grow on complete medium containing 0.3 M CaCl(2). The double msg5 ptc2 disruptant failed to grow on medium containing 1.0 M NaCl and, like the ynr022c single deletion, also could not grow on medium containing 0.3 M CaCl(2). The synthetic phenotypes in the two latter cases where each of the PPases is categorized in a different phosphatase family led us to discuss the novel mechanism involved in the functional redundancy of the PPases.

Calcium Chloride↗