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In JoVE (1)

Other Publications (15)

Articles by Yo Suzuki in JoVE

 JoVE Biology

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

1Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, 2Department of Microbial and Environmental Genomics, J. Craig Venter Institute, 3Donnelly Centre & Department of Molecular Genetics, University of Toronto, 4Lunenfeld Research Institute, Mt Sinai Hospital

JoVE 4072

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

Other articles by Yo Suzuki on PubMed

A Caenorhabditis Elegans TGF-beta, DBL-1, Controls the Expression of LON-1, a PR-related Protein, That Regulates Polyploidization and Body Length

Using cDNA-based array analysis combined with double-stranded RNA interference (dsRNAi), we have identified yk298h6 as a target gene of Caenorhabditis elegans TGF-beta signaling. Worms overexpressing dbl-1, a TGF-beta ligand, are 16% longer than wild type. Array analysis shows yk298h6 to be one of several genes suppressed in such worms. Disruption of yk298h6 function by dsRNAi also resulted in long worms, suggesting that it is a negative regulator of body length. yk298h6 was then mapped to, and shown to be identical to, lon-1, a known gene that affects body length. lon-1 encodes a 312 amino acid protein with a motif sequence that is conserved from plants to humans. Expression studies confirm that LON-1 is repressed by DBL-1, suggesting that LON-1 is a novel downstream component of the C.elegans TGF-beta growth regulation pathway. Consistent with this, LON-1 is expressed mainly in the larval and adult hypodermis and has dose-dependent effects on body length associated with changes in hypodermal ploidy, but not hypodermal cell proliferation.

A Cuticle Collagen Encoded by the Lon-3 Gene May Be a Target of TGF-beta Signaling in Determining Caenorhabditis Elegans Body Shape

The signaling pathway initiated by the TGF-beta family member DBL-1 in Caenorhabditis elegans controls body shape in a dose-dependent manner. Loss-of-function (lf) mutations in the dbl-1 gene cause a short, small body (Sma phenotype), whereas overexpression of dbl-1 causes a long body (Lon phenotype). To understand the cellular mechanisms underlying these phenotypes, we have isolated suppressors of the Sma phenotype resulting from a dbl-1(lf) mutation. Two of these suppressors are mutations in the lon-3 gene, of which four additional alleles are known. We show that lon-3 encodes a collagen that is a component of the C. elegans cuticle. Genetic and reporter-gene expression analyses suggest that lon-3 is involved in determination of body shape and is post-transcriptionally regulated by the dbl-1 pathway. These results support the possibility that TGF-beta signaling controls C. elegans body shape by regulating cuticle composition.

Expression of the C. Elegans Labial Orthologue Ceh-13 During Male Tail Morphogenesis

Hox genes are transcriptional regulators of metazoan body regionalization along the anteroposterior axis that act by specifying positional identity in differentiating cells. ceh-13, the labial orthologue in Caenorhabditis elegans, is expressed both during embryogenesis and post- embryonic development. Using GFP reporter analysis and immunocytochemistry, we discovered a spatiotemporal pattern of gene expression in the male tail during the L3 and L4 larval stages that is TGF-beta pathway-dependent. Analysis of reporter activity in transgenic animals identified a distinct promoter region driving male tail-specific ceh-13 expression. We also report the interspecies conservation of sequence motifs within this region and speculate that, in the course of evolutionary diversification, ceh-13 may have acquired new functionality while conserving its homeotic role.

Genetic Redundancy Masks Diverse Functions of the Tumor Suppressor Gene PTEN During C. Elegans Development

Genetic redundancy is associated with a large percentage of genes. We investigated PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor gene functions that eluded single mutant analyses, using a Caenorhabditis elegans genome-wide screen. We show that at least 27 genes collaborate with the worm PTEN homolog daf-18 for various functions previously concealed by genetic redundancy, including embryogenesis, cuticle turnover, egg laying, and oocyte maturation. In one example, daf-18 appears to constitute a cell-autonomous germline signal that converges with a somatic gonad signal mediated by ceh-18 at a kinase inhibition. We provide evidence that daf-18 elicits some functions independent of the downstream gene daf-16.

Dietary 5-campestenone (campest-5-en-3-one) Enhances Fatty Acid Oxidation in Perfused Rat Liver

The effect of dietary 5-campestenone (campest-5-en-3-one), a chemical modification product of a naturally-occurring plant sterol, campesterol, on lipid metabolism was examined using a rat liver perfusion system. Male Sprague-Dawley rats weighing about 140 g were fed a diet supplemented with or without 0.2% 5-campestenone for 14 d. 5-Campestenone feeding resulted in a marked reduction in the concentrations of serum lipids, such as triacylglycerol (TG), cholesterol, phospholipid, and free fatty acid, without influencing food intake or growth. Then, isolated livers from both groups were perfused for 4 h in the presence of an exogenous linoelaidic acid substrate. Dietary 5-campestenone markedly elevated hepatic ketone body production, while cumulative secretions of TG, cholesterol, and phospholipid by the livers of rats fed 5-campestenone were all significantly lowered as compared to those fed without the compound: the extent of the reduction was more prominent in the secretion of TG than other lipid components. In addition, the reduction of TG secretion was concomitantly accompanied by the reduced incorporation of both exogenous and endogenous fatty acids into this lipid molecule. These results suggest that dietary 5-campestenone exerts its hypotriglyceridemic effect, at least, in part through an enhanced metabolism of endogenous and exogenous fatty acids to oxidation at the expense of esterification in rat liver.

Systematic Genetics Swims Forward Elegantly

Estimation of Interaural Level Difference Based on Anthropometry and Its Effect on Sound Localization

Individualization of head-related transfer functions (HRTFs) is important for highly accurate sound localization systems such as virtual auditory displays. A method to estimate interaural level differences (ILDs) from a listener's anthropometry is presented in this paper to avoid the burden of direct measurement of HRTFs. The main result presented in this paper is that localization is improved with nonindividualized HRTF if ILD is fitted to the listener. First, the relationship between ILDs and the anthropometric parameters was analyzed using multiple regression analysis. The azimuthal variation of the ILDs in each 1/3-octave band was then estimated from the listener's anthropometric parameters. A psychoacoustical experiment was carried out to evaluate its effectiveness. The experimental results show that the adjustment of the frequency characteristics of ILDs for a listener with the proposed method is effective for localization accuracy.

Effect of Dietary Alpha-linolenic Acid on Vascular Reactivity in Aorta of Spontaneously Hypertensive Rats

Several studies of hypertension have shown that dietary alpha-linolenic acid (ALA) has a blood pressure-lowering effect. In the present study, we investigated the effect of dietary ALA on endothelium-dependent vascular reactivity in six-week-old spontaneously hypertensive rats (SHR). At the end of the feeding period, systolic blood pressure in the ALA group was significantly lower than in the control group. There was no significant difference in endothelium-dependent vasorelaxant response between the two diet groups. There was also no significant inter-group difference in the vascular wall thickness and media-to-lumen ratio. In conclusion, the present findings suggest that the blood pressure-lowering mechanism of dietary ALA does not involve increased sensitivity to NO or a change the in vascular morphology of the aorta.

Dietary Alpha-linolenic Acid Inhibits Angiotensin-converting Enzyme Activity and MRNA Expression Levels in the Aorta of Spontaneously Hypertensive Rats

Several studies have shown that dietary alpha-linolenic acid (ALA) is associated with a reduced risk of cardiovascular disease and has an antihypertensive effect. Blood pressure is regulated mainly by angiotensin-converting enzyme (ACE). In the present study, we investigated the effect of dietary ALA on ACE to clarify the mechanism of the antihypertensive effect in spontaneously hypertensive rats (SHR). Six-week-old SHR were fed a diet containing either 10% ALA-rich flaxseed oil or high oleic safflower oil as a control for four weeks. Systolic blood pressure (SBP) was measured by the tail cuff method once weekly. At the end of the feeding period, ACE activity was determined in the heart, aorta, lung and kidney. ACE mRNA in these organs was also measured by real-time PCR analysis. SBP in the ALA group was significantly lower than in the control group at 2, 3, and 4 weeks. The ACE activity and mRNA expression levels in the ALA group were significantly lower than in the control only in the aorta. In conclusion, the present findings suggest that the blood pressure-lowering mechanism of dietary ALA may be involved in the reduction of ACE activity and mRNA expression levels in the aorta of SHR.

Effects of Semen Extenders and Storage Temperatures on Characteristics of Frozen-thawed Bryde's (Balaenoptera Edeni) Whale Spermatozoa

The present study investigated effects of three semen extenders and storage temperatures on post-thaw characteristics of Bryde's whale spermatozoa. Spermatozoa were collected from the vasa deferens of three mature Bryde's whales captured during the Japanese whale research in the north-west Pacific (May to August 2007) after death. The three semen extenders used for freezing were 1) a commercialized synthetic extender (AndroMed: AM), 2) Tris-based + 10% bovine serum albumin (BSA) and 3) Tris-based + egg yolk (EY). The sperm samples from the three whales were frozen with the three extenders, and the post-thaw spermatozoa were stored at three different temperatures (35 C; 20-25 C, room temperature; and 5 C) for 0, 6, 12, 24, 48 and 96 h. At each time-point, total and progressive motility (PM), viability (live or dead), the hypo-osmotic test, defective acrosomes and malformation were examined. Immediately after thawing, AM resulted in similar recovery rates (60.4 and 83.3%) in 2 of the 3 whales examined and had comparable post-thaw recovery rates to those obtained using the EY and BSA extenders. Immediately after thawing, the proportion of PM in EY (17.6%) was higher (P<0.05) than that in BSA (15.0%). In the hypo-osmotic test, the proportions of AM (26.0%) and BSA (25.2%) were higher (P<0.05) than that of EY (17.3 %). The three extenders had similar viabilities (36.7, 37.9 and 32.1%, respectively), but the viability of BSA was higher (P<0.05) than that of EY. The present study showed that a synthetic semen extender, AndroMed, could be used for cryopreservation of whale spermatozoa in addition to Tris-based extenders containing bovine serum albumin or egg yolk. Storage of the post-thaw Bryde's whale spermatozoa was better at 5 C than at room temperature or 35 C. The frozen-thawed Bryde's whale spermatozoa maintained their motility and viability for at least two days at room temperature and for four days at 5 C.

Production of Sei Whale (Balaenoptera Borealis) Cloned Embryos by Inter- and Intra-species Somatic Cell Nuclear Transfer

The objectives of this study were to choose an effective embryo reconstruction method and an effective post-activation agent for in vitro production of sei whale (Balaenoptera borealis) interspecies somatic cell nuclear transfer (iSCNT) embryos. Moreover, trichostatin A (TSA) treatment of whale iSCNT embryos was performed to improve the in vitro embryo development. In Experiment 1, the fusion rate was significantly higher (88.1%) in embryos reconstructed using the intracytoplasmic cell injection method (ICI) than that (48.7%) in the subzonal cell insertion (SUZI) counterpart. The rates of pseudopronucleus (PPN) formation (77.4 vs. 77.2%) and cleavage (24.5 vs. 37.0%) did not vary between ICI and SUZI. However, the PPN formation and cleavage rates were significantly (P<0.05) lower in the iSCNT embryos than in the parthenogenetic control (95.7% and 64.4%, respectively). Although 21.5% of the bovine parthenogenetic embryos developed to the blastocyst stage, no iSCNT embryo developed beyond the 6-cell stage. In Experiment 2, the cleavage rate did not vary between the TSA (50 nM)-treated and non-treated whale iSCNT embryos (30.5 vs. 32.3%, respectively). Moreover, it did not vary between the TSA-treated iSCNT and SCNT embryos (30.5 vs. 32.0%, respectively). Only one TSA non-treated iSCNT embryo developed to a compacted morula with 20 nuclei. One TSA-treated whale SCNT embryo developed to the 8-cell stage, and out of five whale iSCNT embryos, a 6-cell stage embryo was positive for whale DNA. In conclusion, bovine oocytes have the ability to support development of sei whale nuclei up to the 6-cell stage.

Effects of Seasonal Changes on in Vitro Developmental Competence of Porcine Oocytes

The aim of this study was to investigate whether seasonal changes affected in vitro developmental competence of porcine oocytes. The relationship between atmospheric temperature and embryonic development of in vitro matured porcine oocytes following intracytoplasmic sperm injection was examined throughout the year. The blastocyst rate (31.1%) in winter (mean atmospheric temperature during December to February: -3.8 C) was significantly higher (P<0.05) than those of other seasons in 2008/2009 (19.7-23.5%; 6.3-17.5 C). The monthly mean blastocyst rates were negatively correlated with the temperatures (r=-0.5944, P<0.05). The results of the present study suggest that porcine embryos could be produced throughout the year, but the in vitro production efficiency was significantly affected by season, i.e., atmospheric temperatures. Furthermore, the results showed that winter is a favorable season for blastocyst production in the region of Obihiro, Hokkaido, Japan.

Knocking out Multigene Redundancies Via Cycles of Sexual Assortment and Fluorescence Selection

Phenotypes that might otherwise reveal a gene's function can be obscured by genes with overlapping function. This phenomenon is best known within gene families, in which an important shared function may only be revealed by mutating all family members. Here we describe the 'green monster' technology that enables precise deletion of many genes. In this method, a population of deletion strains with each deletion marked by an inducible green fluorescent protein reporter gene, is subjected to repeated rounds of mating, meiosis and flow-cytometric enrichment. This results in the aggregation of multiple deletion loci in single cells. The green monster strategy is potentially applicable to assembling other engineered alterations in any species with sex or alternative means of allelic assortment. To test the technology, we generated a single broadly drug-sensitive strain of Saccharomyces cerevisiae bearing precise deletions of all 16 ATP-binding cassette transporters within clades associated with multidrug resistance.

Reconstitution of Human RNA Interference in Budding Yeast

Although RNA-mediated interference (RNAi) is a widely conserved process among eukaryotes, including many fungi, it is absent from the budding yeast Saccharomyces cerevisiae. Three human proteins, Ago2, Dicer and TRBP, are sufficient for reconstituting the RISC complex in vitro. To examine whether the introduction of human RNAi genes can reconstitute RNAi in S. cerevisiae, genes encoding these three human proteins were introduced into S. cerevisiae. We observed both siRNA and siRNA- and RISC-dependent silencing of the target gene GFP. Thus, human Ago2, Dicer and TRBP can functionally reconstitute human RNAi in S. cerevisiae, in vivo, enabling the study and use of the human RNAi pathway in a facile genetic model organism.

Systematic Exploration of Synergistic Drug Pairs

Drug synergy allows a therapeutic effect to be achieved with lower doses of component drugs. Drug synergy can result when drugs target the products of genes that act in parallel pathways ('specific synergy'). Such cases of drug synergy should tend to correspond to synergistic genetic interaction between the corresponding target genes. Alternatively, 'promiscuous synergy' can arise when one drug non-specifically increases the effects of many other drugs, for example, by increased bioavailability. To assess the relative abundance of these drug synergy types, we examined 200 pairs of antifungal drugs in S. cerevisiae. We found 38 antifungal synergies, 37 of which were novel. While 14 cases of drug synergy corresponded to genetic interaction, 92% of the synergies we discovered involved only six frequently synergistic drugs. Although promiscuity of four drugs can be explained under the bioavailability model, the promiscuity of Tacrolimus and Pentamidine was completely unexpected. While many drug synergies correspond to genetic interactions, the majority of drug synergies appear to result from non-specific promiscuous synergy.

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