Takahiko Utsugi

Movement of yeast 1,3‐β‐glucan synthase is essential for uniform cell wall synthesis

Background:  The cell wall has an important role in maintaining cell shape. In the budding yeast Saccharomyces cerevisiae, the major filamentous component of the cell wall responsible for its rigidity is 1,3‐β‐glucan and is synthesized by 1,3‐β‐glucan synthase (GS), localized on the plasma membrane.

Isolation and characterization of cDNA clones encodingcdc2 homologues fromOryza sativa: a functional homologue and cognate variants

SummaryUsing probes obtained by PCR amplification, we have isolated two cognate rice cDNAs (cdc2Os-1 andcdc2Os-2) encoding structural homologues of thecdc2+/CDC28(cdc2) protein kinase from a cDNA library prepared from cultured rice cells. Comparison of the deduced amino acid sequences of cdc2Os-1 and cdc2Os-2 showed that they are 83 % identical. They are 62 % identical toCDC28 ofSaccharomyces cerevisiae and much more similar to the yeast and mammalian p34cdc2 kinases than to riceR2, acdc2-related kinase isolated previously by screening the same rice cDNA library with a different oligonucleotide probe. Southern blot analysis indicated that the three rice clones (cdc2Os-1,cdc2Os-2 andR2) are derived from distinct genes and are each found in a single copy per rice haploid genome. RNA blot analysis revealed that these genes are expressed in proliferating rice cells and in young rice seedlings.cdc2Os-1 could complement a temperature-sensitive yeast mutant ofcdc28. However, despite the similarity in structure, bothcdc2Os-2 andR2 were unable to complement the same mutant. Thus, the present results demonstrate the presence of structurally related, but functionally distinct cognates of thecdc2 cell cycle kinase in rice.

Dynactin is involved in a checkpoint to monitor cell wall synthesis in Saccharomyces cerevisiae

Checkpoint controls ensure the completion of cell cycle events with high fidelity in the correct order. Here we show the existence of a novel checkpoint that ensures coupling of cell wall synthesis and mitosis. In response to a defect in cell wall synthesis, S. cerevisiae cells arrest the cell-cycle before spindle pole body separation. This arrest results from the regulation of the M-phase cyclin Clb2p at the transcriptional level through the transcription factor Fkh2p. Components of the dynactin complex are required to achieve the G2 arrest whilst keeping cells highly viable. Thus, the dynactin complex has a function in a checkpoint that monitors cell wall synthesis.

Yeast tom1 mutant exhibits pleiotropic defects in nuclear division, maintenance of nuclear structure and nucleocytoplasmic transport at high temperatures

A tom1-1 mutant was isolated from Saccharomyces cerevisiae. At high temperatures, 60% of the cells were arrested as dumbbell forms with a single large nucleus containing duplicated DNA and a short spindle. Electron-microscopy showed electron-dense structures scattered within the nucleus. Indirect immunofluorescent microscopy revealed these structures to be fragmented nucleoli since the dotted structures were stained with anti-Nop1(fibrillarin) antibody in large regions of the nuclei. Fluorescent in situ hybridization analysis using oligo(dT) revealed nuclear accumulation of poly(A)+RNA. We cloned TOM1 which encodes a large protein (380kDa) with a hect (homologous to E6-AP C terminus)-domain at its C terminus. Deletions of either this hect-region or the entire gene made cellular growth temperature-sensitive. Site-directed mutagenesis of the conserved cysteine residue (tom1C3235A) in the hect-domain, supposed to be necessary for thioester-bond formation with ubiquitin, abolished the gene function. When a functional glutathione S-transferase (GST)-tagged hect protein was overproduced, it facilitated the protein conjugation with a myc-tagged ubiquitinRA, while this was not seen when GST-hectC3235A was overproduced. The protein conjugation with a hemagglutinin-tagged Smt3 was not affected by the overproduction of GST-hect. Taken together, we suggest that Tom1 is a ubiquitin ligase. As a multi-copy suppressor of tom1, we isolated STM3/NPI46/FPR3 which encodes a nucleolar nucleolin-like protein. We discuss possible functions of Tom1 with respect to the pleiotropic defects of nuclear division, maintenance of nuclear structure, and nucleocytoplasmic transport.

A high dose of theSTM1 gene suppresses the temperature sensitivity of thetom1 andhtr1 mutants inSaccharomyces cerevisiae

Abstract A new gene ( STM1 ;_suppressor of_to_ml) Saccharomyces cerevisiae was isolated by the ability to suppress the temperature sensitivity of a tom1 mutant, by increasing its gene dosage. The gene could also suppress the temperature sensitivity of the htr1 disruptant (Kikuchi et al. (1994) Mol. Gen. Genet. 245, 107–116) and was physically mapped in the region near PEP3 on chromosome XII R. The predicted gene product (29999 Da) is basic and partially homologous to various histone H1. The level of the gene expression increased 2-fold when exposed to mating pheromone.

Novel DNA Microarray System for Analysis of Nascent mRNAs

Transcriptional activation and repression are a key step in the regulation of all cellular activities. The development of comprehensive analysis methods such as DNA microarray has advanced our understanding of the correlation between the regulation of transcription and that of cellular mechanisms. However, DNA microarray analysis based on steady-state mRNA (total mRNA) does not always correspond to transcriptional activation or repression. To comprehend these transcriptional regulations, the detection of nascent RNAs is more informative. Although the nuclear run-on assay can detect nascent RNAs, it has not been fully applied to DNA microarray analysis. In this study, we have developed a highly efficient method for isolating bromouridine-labeled nascent RNAs that can be successfully applied to DNA microarray analysis. This method can linearly amplify small amounts of mRNAs with little bias. Furthermore, we have applied this method to DNA microarray analysis from mouse G2-arrested cells and have identified several genes that exhibit novel expression profiles. This method will provide important information in the field of transcriptome analysis of various cellular processes.

Genetic analyses using a mouse cell cycle mutant identifies magoh as a novel gene involved in Cdk regulation.

Many of the genes that control cyclin‐dependent kinase (Cdks) activity have been identified by genetic research using yeast mutants. Suppression analysis and synthetic enhancement analysis are two broad approaches to the identification of genetic interaction networks in yeasts. Here we show, by genetic analyses using a mammalian cell cycle mutant, that mouse magoh is involved in Cdk regulation. Magoh, a homolog of the Drosophila mago nashi gene product, is a component of the splicing‐dependent exon–exon junction complex (EJC). We show that, in addition to ccnb1 and cks2, magoh is also a dosage suppressor of the mouse temperature‐sensitive cdc2 mutant, and synthetic enhancement of the cdc2 ts phenotype by RNA interference (RNAi) of magoh is observed in a manner similar to RNAi of cks2. Moreover, the depletion of magoh by RNAi causes cold‐sensitive defects in the cell cycle transition, and exogenous cks2 expression partially suppresses the defect. Consistent with the genetic evidence, magoh RNAi caused defects in the expression of Cdc2 or Cks proteins, and introns of cks genes strongly affected protein expression levels. Thus, these data suggest that mouse Magoh is related to cell cycle regulation.

Phosphorylation of human INO80 is involved in DNA damage tolerance

Double strand breaks (DSBs) are the most serious type of DNA damage. DSBs can be generated directly by exposure to ionizing radiation or indirectly by replication fork collapse. The DNA damage tolerance pathway, which is conserved from bacteria to humans, prevents this collapse by overcoming replication blockages. The INO80 chromatin remodeling complex plays an important role in the DNA damage response. The yeast INO80 complex participates in the DNA damage tolerance pathway. The mechanisms regulating yINO80 complex are not fully understood, but yeast INO80 complex are necessary for efficient proliferating cell nuclear antigen (PCNA) ubiquitination and for recruitment of Rad18 to replication forks. In contrast, the function of the mammalian INO80 complex in DNA damage tolerance is less clear. Here, we show that human INO80 was necessary for PCNA ubiquitination and recruitment of Rad18 to DNA damage sites. Moreover, the C-terminal region of human INO80 was phosphorylated, and overexpression of a phosphorylation-deficient mutant of human INO80 resulted in decreased ubiquitination of PCNA during DNA replication. These results suggest that the human INO80 complex, like the yeast complex, was involved in the DNA damage tolerance pathway and that phosphorylation of human INO80 was involved in the DNA damage tolerance pathway. These findings provide new insights into the DNA damage tolerance pathway in mammalian cells.

α-1,6-Fucosyltransferase (FUT8) inhibits hemoglobin production during differentiation of murine and K562 human erythroleukemia cells.

Background: The overall view of erythropoiesis remains unclear. Results: Overexpression of α-1,6-fucosyltransferase inhibits hemoglobin production in murine and human erythroleukemia cells; down-regulation of α-1,6-fucosyltransferase promotes hemoglobin production and erythroid differentiation of human erythroleukemia cells. Conclusion: Core fucosylation plays an important role in hemoglobin production and erythroid differentiation. Significance: This might be the first finding that glycosylation negatively regulates erythroid differentiation. Erythropoiesis results from a complex combination of the expression of several transcription factor genes and cytokine signaling. However, the overall view of erythroid differentiation remains unclear. First, we screened for erythroid differentiation-related genes by comparing the expression profiles of high differentiation-inducible and low differentiation-inducible murine erythroleukemia cells. We identified that overexpression of α-1,6-fucosyltransferase (Fut8) inhibits hemoglobin production. FUT8 catalyzes the transfer of a fucose residue to N-linked oligosaccharides on glycoproteins via an α-1,6 linkage, leading to core fucosylation in mammals. Expression of Fut8 was down-regulated during chemically induced differentiation of murine erythroleukemia cells. Additionally, expression of Fut8 was positively regulated by c-Myc and c-Myb, which are known as suppressors of erythroid differentiation. Second, we found that FUT8 is the only fucosyltransferase family member that inhibits hemoglobin production. Functional analysis of FUT8 revealed that the donor substrate-binding domain and a flexible loop play essential roles in inhibition of hemoglobin production. This result clearly demonstrates that core fucosylation inhibits hemoglobin production. Third, FUT8 also inhibited hemoglobin production of human erythroleukemia K562 cells. Finally, a short hairpin RNA study showed that FUT8 down-regulation induced hemoglobin production and increase of transferrin receptor/glycophorin A-positive cells in human erythroleukemia K562 cells. Our findings define FUT8 as a novel factor for hemoglobin production and demonstrate that core fucosylation plays an important role in erythroid differentiation.

Identification of preferentially reactivated genes during early G1 phase using nascent mRNA as an index of transcriptional activity.

During mammalian mitosis, transcription is silenced due to dissociation of transcription factors from DNA and chromosome condensation. At the end of mitosis, transcription is reactivated through chromosome relaxation and reloading of these factors to the DNA. Early G1 genes, which are preferentially reactivated during M/G1 transition, are important for maintenance of cellular function and are known to be strictly regulated. As only few early G1 genes have been identified to date, screening for early G1 genes by genome-wide analysis using nascent mRNA could contribute to the elucidation of the regulatory mechanisms during early G1. Here, we performed a detailed expression analysis for the M/G1 transition of mammalian cells by microarray analysis of nascent mRNA, and identified 298 early G1 genes. Analysis of these genes provides two important insights. Firstly, certain motifs are enriched in the upstream sequences of early G1 genes; from this we could predict candidate cognate transcription factors, including Sp1, which is recruited to the DNA in the early G1 phase. Secondly, we discovered that neighboring genes of early G1 genes were also frequently up-regulated in the G1 phase. Information about these numerous newly identified early G1 genes will likely contribute to an understanding of the regulatory mechanisms of the early G1 genes.