Mutation Analysis of Synthetic DNA Barcodes in a Fission Yeast Gene Deletion Library by Sanger Sequencing.

Incorporation of unique barcodes into fission yeast gene deletion collections has enabled the identification of gene functions by growth fitness analysis. For fine tuning, it is important to examine barcode sequences, because mutations arise during strain construction. Out of 8,708 barcodes (4,354 strains) covering 88.5% of all 4,919 open reading frames, 7,734 barcodes (88.8%) were validated as high-fidelity to be inserted at the correct positions by Sanger sequencing. Sequence examination of the 7,734 high-fidelity barcodes revealed that 1,039 barcodes (13.4%) deviated from the original design. In total, 1,284 mutations (mutation rate of 16.6%) exist within the 1,039 mutated barcodes, which is comparable to budding yeast (18%). When the type of mutation was considered, substitutions accounted for 845 mutations (10.9%), deletions accounted for 319 mutations (4.1%), and insertions accounted for 121 mutations (1.6%). Peculiarly, the frequency of substitutions (67.6%) was unexpectedly higher than in budding yeast (∼28%) and well above the predicted error of Sanger sequencing (∼2%), which might have arisen during the solid-phase oligonucleotide synthesis and PCR amplification of the barcodes during strain construction. When the mutation rate was analyzed by position within 20-mer barcodes using the 1,284 mutations from the 7,734 sequenced barcodes, there was no significant difference between up-tags and down-tags at a given position. The mutation frequency at a given position was similar at most positions, ranging from 0.4% (32/7,734) to 1.1% (82/7,734), except at position 1, which was highest (3.1%), as in budding yeast. Together, well-defined barcode sequences, combined with the next-generation sequencing platform, promise to make the fission yeast gene deletion library a powerful tool for understanding gene function.

NSC126188, a piperazine alkyl derivative, induces apoptosis via upregulation of RhoB in HeLa cells.

We describe here a piperazine alkyl derivative, NSC126188, which induced apoptosis of HeLa cells by upregulating RhoB expression. NSC126188 caused multi-septation of fission yeast and hypersensitized a ∆rho3 mutant, which implicates the involvement of functional human homolog RhoB. The treatment of cells with NSC126188 induced apoptosis and a dramatic increase in RhoB expression. In addition, RhoB knockdown using siRNA rescued cells from apoptosis, indicating a crucial role of RhoB in NSC126188-induced apoptosis. In a reporter assay using luciferase and EGFP under control of the RhoB promoter, NSC126188 increased both luciferase activity and the expression of EGFP, implicating transcriptional activation of RhoB by NSC126188. Furthermore, NSC126188 demonstrated in vivo anti-tumor activity, inhibiting tumor growth by 66.8% in a nude mouse xenograft using PC-3 human prostate cancer cells. These results suggest that NSC126188 is a potential lead compound and that upregulation of RhoB is associated with NSC126188-induced apoptosis.

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.

RhoB induces apoptosis via direct interaction with TNFAIP1 in HeLa cells.

RhoB, a tumor suppressor, has emerged as an interesting cancer target, and extensive studies aimed at understanding its role in apoptosis have been performed. In our study, we investigated the involvement of RhoB-interacting molecules in apoptosis. To identify RhoB-interacting proteins, we performed yeast-two hybrid screening assays using RhoB as a bait and isolated TNFAIP1, a TNFalpha-induced protein containing the BTB/POZ domain. The interaction between RhoB and TNFAIP1 was demonstrated in vivo through coimmunoprecipitation studies and in vitro binding assays. RFP-TNFAIP1 was found to be partially colocalized with EGFP-RhoB. The partial colocalization of RhoB and TNFAIP1 in endosomes suggests that RhoB-TNFAIP1 interactions may have a functional role in apoptosis. TNFAIP1 elicited proapoptotic activity, while simultaneous expression of RhoB and TNFAIP1 resulted in a dramatic increase in apoptosis in HeLa cells. Furthermore, knockdown of RhoB using siRNA clearly rescued cells from apoptosis induced by TNFAIP1. This finding suggests that interactions between RhoB and TNFAIP1 are crucial for induction of apoptosis in HeLa cells. The observation of increased SAPK/JNK phosphorylation in apoptotic cells and the finding that a JNK inhibitor suppressed apoptosis indicates that SAPK/JNK signaling may be involved in apoptosis induced by RhoB-TNFAIP1 interactions. In conclusion, we found that RhoB interacts with TNFAIP1 to regulate apoptosis via a SAPK/JNK-mediated signal transduction mechanism.

Genome-wide drug-induced haploinsufficient screening of fission yeast for identification of hydrazinocurcumin targets.

Hydrazinocurcumin (HC), a synthetic derivative of curcumin, has been reported to inhibit angiogenesis via unknown mechanisms. Understanding the molecular mechanisms of the drug's action is important for the development of improved compounds with better pharmacological properties. A genomewide drug-induced haploinsufficiency screening of fission yeast gene deletion mutants has been applied to identify drug targets of HC. As a first step, the 50% inhibition concentration (IC50) of HC was determined to be 2.2 microM. The initial screening of 4,158 mutants in 384-well plates using robotics was performed at concentrations of 2, 3, and 4 microM. A second screening was performed to detect sensitivity to HC on the plates. The first screening revealed 178 candidates, and the second screening resulted in 13 candidates, following the elimination of 165 false positives. Final filtering of the condition-dependent haploinsufficient genes gave eight target genes. Analysis of the specific targets of HC has shown that they are related to septum formation and the general transcription processes, which may be related to histone acetyl transferase. The target mutants showed 65% growth inhibition in response to HC compared with wild-type controls, as shown by liquid culture assay.

Identification of small molecules inducing apoptosis by cell-based assay using fission yeast deletion mutants.

The cell-based assay using yeast deletion mutants has been recognized as an efficient analysis to discover therapeutic compounds and reveal their mode of action. In this study, S. pombe deletion mutants-based HTS screening was carried out to identify potential anti-cancer agents. The NCI chemical library of 5700 compounds was screened using kit strains, which consisted of S. pombe mutants harboring deletions in genes involved in DNA repair and mitotic control. During the screening, we identified 40 compounds conferring growth inhibition of S. pombe. Their anti-tumorigenic properties were examined by phenotypic effect on S. pombe, flow cytometry and apoptosis analysis of human cancer. Here, we report hit compounds inducing apoptosis for development of anti-cancer agents suggesting that S. pombe deletion mutants are useful in identifying potential anti-cancer agents in human cancer therapeutics.

Rapid screen of human genes for relevance to cancer using fission yeast.

A total of 437 human full-length cDNAs isolated by microarray analysis of liver and/or gastric cancer tissues were evaluated for their relevance to cancer using the fission yeast Schizosaccharomyces pombe. Overexpression of 161 human cDNAs in S. pombe caused growth inhibition and/or morphological changes, which can be considered as cancer-related phenotypes of S. pombe. Sixteen genes causing growth defects and morphological changes at the same time were chosen to validate their ostensible oncogenic properties. They were highly expressed in liver and/or gastric cancer cell lines. Also, when the mouse embryonic fibroblast cell type NIH3T3 was transfected with these genes, the proliferation rates of cells were increased by 32% to 120%. This study demonstrates that fission yeast can be used as an advantageous and powerful tool for the rapid screening of human genes relevant to cancer. Furthermore, the human genes screened can be tested further as diagnostic markers and potential therapeutic targets for liver and stomach cancers. They also can be studied further for the elucidation of mechanisms involved in carcinogenesis.

Cerulenin-mediated apoptosis is involved in adenine metabolic pathway.

Cerulenin, a fatty acid synthase (FAS) inhibitor, induces apoptosis of variety of tumor cells. To elucidate mode of action by cerulenin, we employed the proteomics approach using Schizosaccharomyces pombe. The differential protein expression profile of S. pombe revealed that cerulenin modulated the expressions of proteins involved in stresses and metabolism, including both ade10 and adk1 proteins. The nutrient supplementation assay demonstrated that cerulenin affected enzymatic steps transferring a phosphoribosyl group. This result suggests that cerulenin accumulates AMP and p-ribosyl-s-amino-imidazole carboxamide (AICAR) and reduces other necessary nucleotides, which induces feedback inhibition of enzymes and the transcriptional regulation of related genes in de novo and salvage adenine metabolic pathway. Furthermore, the deregulation of adenine nucleotide synthesis may interfere ribonucleotide reductase and cause defects in cell cycle progression and chromosome segregation. In conclusion, cerulenin induces apoptosis through deregulation of adenine nucleotide biosynthesis resulting in nuclear division defects in S. pombe.

Systematic identification of hepatocellular proteins interacting with NS5A of the hepatitis C virus.

The hepatitis C virus is associated with the development of liver cirrhosis and hepatocellular carcinomas. Among the 10 polyproteins produced by the virus, no function has been clearly assigned to the non-structural 5A (NS5A) protein. This study was designed to identify the hepatocellular proteins that interact with NS5A of the HCV. Yeast two-hybrid experiments were performed with a human liver cDNA prey-library, using five different NS5A derivatives as baits, the full-length NS5A (NS5A-F, amino acid (aa) 1 approximately 447) and its four different derivatives, denoted as NS5A-A (aa 1 approximately 150), -B (aa 1 approximately 300), -C (aa 300 approximately 447) and D (aa 150 approximately 447). NS5A-F, NS5A-B and NS5A-C gave two, two and 10 candidate clones, respectively, including an AHNAK-related protein, the secreted frizzled-related protein 4 (SFRP4), the N-myc downstream regulated gene 1 (NDRG1), the cellular retinoic acid binding protein 1 (CRABP-1), ferritin heavy chain (FTH1), translokin, tumor-associated calcium signal transducer 2 (TACSTD2), phosphatidylinositol 4-kinase (PI4K) and centaurindelta 2 (CENTdelta2). However, NS5A-A produced no candidates and NS5A-D was not suitable as bait due to transcriptional activity. Based on an in vitro binding assay, CRABP-1, PI4K, CENTdelta2 and two unknown fusion proteins with maltose binding protein (MBP), were confirmed to interact with the glutathione S-transferase (GST)/NS5A fusion protein. Furthermore, the interactions of CRABP-1, PI4K and CENTdelta2 were not related to the PXXP motif (class II), as judged by a domain analysis. While their biological relevance is under investigation, the results contribute to a better understanding of the possible role of NS5A in hepatocellular signaling pathways.

Electrochemical sensor for electrochemically inactive beta-D(+)-glucose using alpha-cyclodextrin template molecules.

We report an electrochemical sensor for an electrochemically inactive organic compound using a self-assembled monolayer (SAM) formed on the gold surface from a solution of thiolated alpha-cyclodextrin (alpha-CD). The SAM makes up an array of ultramicroelectrodes, which capture electroactive molecules such as those of ferrocene. When this SAM-modified electrode is exposed to a solution containing an electrochemically inactive compound, e.g., glucose, the captured ferrocene molecules are replaced by the electroinactive molecules via an equilibrium established between the two compounds, lowering the current for ferrocene oxidation. The decrease in current is directly proportional to the amount of glucose added and the glucose can be analyzed up to approximately 0.80 mM, which is approximately 1/10 of a typical physiological concentration in blood serum. Formation constants of the surface-bound alpha-CD molecules with ferrocene and glucose are also reported.