Hyong Bai Kim

Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe.

We report the construction and analysis of 4,836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast genome providing a tool for studying eukaryotic biology. Comprehensive gene dispensability comparisons with budding yeast—the only other eukaryote for which a comprehensive knockout library exists—revealed that 83% of single-copy orthologs in the two yeasts had conserved dispensability. Gene dispensability differed for certain pathways between the two yeasts, including mitochondrial translation and cell cycle checkpoint control. We show that fission yeast has more essential genes than budding yeast and that essential genes are more likely than nonessential genes to be present in a single copy, to be broadly conserved and to contain introns. Growth fitness analyses determined sets of haploinsufficient and haploproficient genes for fission yeast, and comparisons with budding yeast identified specific ribosomal proteins and RNA polymerase subunits, which may act more generally to regulate eukaryotic cell growth.

Accurate guanine:cytosine discrimination in T4 DNA ligase-based single nucleotide polymorphism analysis using an oxanine-containing ligation fragment.

T4 DNA ligase-based mismatch detection methods have been proposed as useful strategies for single nucleotide polymorphism (SNP) analyses. However, there is a critical problem for cytosine/thymine (C/T) SNP analyses: guanine:thymine (G:T) mismatch is not distinguished from guanine:cytosine (G:C). Here we employed chemically modified nucleobases, such as oxanine and hypoxanthine, at the end of a ligation fragment and analyzed their influence on the ligation efficiency between G:C and G:T. Successful ligation for G:C and no ligation for G:T were observed when oxanine was employed adjacent to guanine in the ligation junction. This ligation method using an oxanine-containing fragment has strong potentials for the accurate analysis of C/T SNPs.

Mad1p, a component of the spindle assembly checkpoint in fission yeast, suppresses a novel septation-defective mutant, sun1, in a cell-division cycle.

To enhance our understanding of the cytokinesis, we have carried out a genetic screen for temperature-sensitive Schizosaccharomyces pombe mutants that show defects in septum formation and cell division. Here we present the isolation and characterization of a new temperature-sensitive mutant, sun1 (septum uncontrolled), which undergoes uncontrolled septation during cell-division cycle at restrictive temperature (37 degrees C). In sun1 mutant, the actin ring and septum are positioned at random locations and angles, and the nuclear division cycle continues. These observations suggest that the sun1 gene product is required for the proper placement of the actin ring as well as precise septation. In a screen for the sun1(+) gene to complement the sun1 mutant, we have isolated a mad1(+) (mitotic arrest deficient) gene, which encodes a component of the spindle checkpoint in the cell-division cycle. Analysis of crossing the sun1 cell with the mad1(+) null mutant indicates that mad1(+) suppresses the sun1 mutant defective in controlled septation in a cell-division cycle.

Molecular Cloning and High-Level Expression of G2 Protein of Hantaan (HTN) Virus 76–118 Strain in the Yeast Pichia Pastoris KM71

Hantaan viral G2 envelope gene, which is known to be one of major antigens and induce neutralizing antibodies, was cloned into expression vector pHIL-S1 which consists of AOX1 promoter, PHO1 signal sequence, HIS4 gene and other components. The recombined plasmid was transformed into methylotropic yeast, Pichia pastoris of KM71 and recombinant strains harboring multi-copy of G2 gene were selected. Expression of the cloned G2 gene was confirmed with Western blot analysis using anti-sera of guinea pig immunized with the carboxyl terminal region of G2 protein expressed in Eschreichia coli. The expression of G2 gene from the recombinant strain was tightly repressed by dextrose and effectively induced by methanol, an inducer of AOX1 promoter. The highest expression level was observed from 1 day after induction and maintained at the same level for up to 4 days.

Highly effective detection of inflamed cells using a modified bradykinin ligand labeled with FITC fluorescence.

Detection of inflammation in live cells is important because long-lasting inflammation is considered to be a primary cause of several diseases. However, few reports have been published on imaging analysis of inflammation in live cells. In this study, we developed an effective imaging system for detection of inflamed cells using a bradykinin ligand (BK) or a modified BK (mBK), which has specific affinity with the cellular B1R receptor. Synthetic BK or mBK labeled with FITC at the N-terminus was employed for discriminating between inflamed and normal cells; this method was found to be effective for detection of inflammation in live cells. In addition, using the mBK-based cell imaging system, we successfully performed flow-based analysis of live cell inflammation on a micro-chip channel, composed of a Starna flow cell and PDMS (Polydimethylsiloxane) walls. The BK-based cell imaging methods designed here would be a useful platform for development of a high-throughput live cell analysis system for investigating the factors underlying inflammation or for screening of anti-inflammation candidate drugs.