GEX1A is a microbial product with antitumor activity. HeLa cells cultured with GEX1A accumulated p27(Kip) and its C-terminally truncated form p27*. GEX1A inhibited the pre-mRNA splicing of p27, producing p27* from the unspliced mRNA containing the first intron. p27* lacked the site required for E3 ligase-mediated proteolysis of p27, leading to its accumulation in GEX1A-treated cells. The accumulated p27* was able to bind to and inhibit the cyclin E-Cdk2 complex that causes E3 ligase-mediated degradation of p27, which probably triggers the accumulation of p27. By using a series of photoaffinity-labeling derivatives of GEX1A, we found that GEX1A targeted SAP155 protein, a subunit of SF3b responsible for pre-mRNA splicing. The linker length between the GEX1A pharmacophore and the photoreactive group was critical for detection of the GEX1A-binding protein. GEX1A serves as a novel splicing inhibitor that specifically impairs the SF3b function by binding to SAP155.

Green synthesis of metal nanoparticles, especially gold nanoparticles (AuNPs), has attracted the great interest of scientists and engineers in the medical and pharmaceutical fields; thus, a variety of ecofriendly, energy-and cost-saving techniques have been developed. In this study, we found that cells of Leptothrix (iron-oxidizing bacteria) released extracellular RNA some of which could exist as a constituent of the cell-enclosing sheaths. As a part of studies of metal encrustation in the sheaths, here we show that RNA prepared from the Leptothrix cells can reduce Au(III) and spherical AuNPs eventually form when an aqueous HAuCl 4 solution is added under ambient conditions. RNA and DNA of other organismal origins have the same ability. Of the nucleosides and nucleobases, only guanosine and guanine can form AuNPs. The DNA moiety, 2′-deoxyguanosine (dG) (used as a reference material), forms AuNPs when mixed with HAuCl 4 solution, but 8-hydroxy-2′-deoxyguanosine (8-OHdG) does not, indicating that AuNP formation evidently depends on the reduction potential of the guanine moiety, not the sugar moiety. This finding is the first demonstration that spherical AuNPs of ca. 5 nm diameter can be obtained by simply adding guanine to HAuCl 4 solution at ambient temperature; no other chemicals or physical treatments are needed.

Sheath-forming, iron-oxidizing bacteria of the genus Leptothrix The genus Leptothrix, which belongs to protobacteria [9], is a group of Gram-negative bacteria [6,10] having a monotrichous, polar, and curvy flagellum [11]. Their rod-shaped cells are relatively large (2.0-3.0 × 0.6-1.0 µm) for bacteria [6,10,11] (L. cholodnii cells are particularly long= ~ 5 µm [8]). Another common physiological character of this genus includes their tendency to form globules of poly-hydroxybutyrate in their cytoplasm as a reserve material, which enables them to survive in nutrient-poor environments [10,11].

Ime1 plays a pivotal role in the initiation of meiosis in a/alpha diploid cells of Saccharomyces cerevisiae. In the absence of glucose and nitrogen, IME1 expression is greater in a/alpha cells than in either a or alpha cells and therefore only a/alpha, but not a/a or alpha/alpha, cells are committed to sporulation. It is known that IME1 expression is positively regulated by Mck1, Rim1, Ime4 and the Swi-Snf complex but other factors may also be involved. In addition, Rme1 is assumed to repress IME1 expression. To provide more details of the repression of expression of IME1, we have isolated mutants in which the IME1p-PHO5 fusion gene integrated at the ura3 locus is expressed in alpha cells under nutritionally rich conditions. We found that mutations occurred in TUP1, SSN6, SIN4 and RGR1, among which TUP1 and SSN6 were identified for the first time as negative regulators of IME1 expression. Deletion of the Rme1-binding site from the IME1 promoter did not result in activation of the expression of IME1 under nutritionally rich conditions, suggesting that Rme1 does not function as a DNA-binding protein with the Tup1-Ssn6 repression complex. We also demonstrated that the 294-bp fragment from nucleotide position -914 to -621 and the 301-bp fragment from nucleotide position -1215 to -915 of the IME1 promoter region contain elements acting as URS and UAS in TUP1+ and tup1 mutant cells, respectively. These findings indicate that IME1 is negatively regulated by the Tup1-Ssn6 repressor complex through two distinct upstream regions in conjunction with unidentified DNA-binding proteins.

The S-phase DNA damage checkpoint is activated by DNA damage to delay DNA synthesis allowing time to resolve the replication block. We previously discovered the p53-dependent S-phase DNA damage checkpoint in mouse zygotes fertilized with irradiated sperm. Here, we report that the same p53 dependency holds in mouse embryonic fibroblasts (MEFs) at low doses of irradiation. DNA synthesis in p53 wild-type (WT) MEFs was suppressed in a biphasic manner in which a sharp decrease below 2.5 Gy was followed by a more moderate decrease up to 10 Gy. In contrast, p53À/À MEFs exhibited radioresistant DNA synthesis below 2.5 Gy whereas the cells retained the moderate suppression above 5 Gy. DNA fiber analysis revealed that 1 Gy irradiation suppressed replication fork progression in p53 WT MEFs, but not in p53À/À MEFs. Proliferating cell nuclear antigen (PCNA), clamp loader of DNA polymerase, was phosphorylated in WT MEFs after 1 Gy irradiation and redistributed to form foci in the nuclei. In contrast, PCNA was not phosphorylated and dissociated from chromatin in 1 Gy-irradiated p53À/À MEFs. These results demonstrate that the novel low-dosespecific p53-dependent S-phase DNA damage checkpoint is likely to regulate the replication fork movement through phosphorylation of PCNA.