Ribonuclease Activity of Dis3 Is Required for Mitotic Progression and Provides a Possible Link between Heterochromatin and Kinetochore Function

BACKGROUND: Cellular RNA metabolism has a broad range of functional aspects in cell growth and division, but its role in chromosome segregation during mitosis is only poorly understood. The Dis3 ribonuclease is a key component of the RNA-processing exosome complex. Previous isolation of the dis3-54 cold-sensitive mutant of fission yeast Schizosaccharomyces pombe suggested that Dis3 is also required for correct chromosome segregation. METHODOLOGY/PRINCIPAL FINDINGS: We show here that the progression of mitosis is arrested in dis3-54, and that segregation of the chromosomes is blocked by activation of the mitotic checkpoint control. This block is dependent on the Mad2 checkpoint protein. Double mutant and inhibitor analyses revealed that Dis3 is required for correct kinetochore formation and function, and that this activity is monitored by the Mad2 checkpoint. Dis3 is a member of the highly conserved RNase II family and is known to be an essential subunit of the exosome complex. The dis3-54 mutation was found to alter the RNaseII domain of Dis3, which caused a reduction in ribonuclease activity in vitro. This was associated with loss of silencing of an ura4(+) reporter gene inserted into the outer repeats (otr) and central core (cnt and imr) regions of the centromere. On the other hand, centromeric siRNA maturation and formation of the RITS RNAi effector complex was normal in the dis3-54 mutant. Micrococcal nuclease assay also suggested the overall chromatin structure of the centromere was not affected in dis3-54 mutant. CONCLUSIONS/SIGNIFICANCE: RNase activity of Dis3, a core subunit of exosome, was found to be required for proper kinetochore formation and establishment of kinetochore-microtubule interactions. Moreover, Dis3 was suggested to contribute to kinetochore formation through an involvement in heterochromatic silencing at both outer centromeric repeats and within the central core region. This activity is likely monitored by the mitotic checkpoint, and distinct from that of RNAi-mediated heterochromatin formation directly targeting outer centromeric repeats

Refined human artificial chromosome vectors for gene therapy and animal transgenesis

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis

Genome evolution in the allotetraploid frog Xenopus laevis

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of ???fossil??? transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.ope

Blunt needle revision with viscoelastic materials via the anterior chamber for early failed filtering blebs after trabeculectomy

Nozomi Kinoshita, Ayumi Ota, Fumihiko Toyoda, Hiroko Yamagami, Akihiro KakehashiDepartment of Ophthalmology, Saitama Medical Center, Jichi Medical University, Saitama, JapanPurpose: To report a new technique of blunt needle revision with viscoelastic materials via the anterior chamber for the treatment of early failed filtering blebs and elevated intraocular pressure after trabeculectomy, in which digital ocular massage and laser suture lysis have been ineffective.Methods: A 27-gauge blunt needle attached to a syringe containing viscoelastic material was inserted into the anterior chamber from the inferior paracentesis. The needle tip was inserted into the subscleral flap space from the filtering fistula at the anterior chamber side, and the scleral flap was lifted bluntly. The needle tip was then inserted into the subconjunctival space where the viscoelastic agent was injected and the adhesion between the sclera and conjunctiva was separated bluntly. Blunt needle revision via the anterior chamber was performed 14 times in six eyes of six patients at Saitama Medical Center, Jichi Medical University from January 2007 to May 2009. All procedures were performed within 1 month after trabeculectomy.Results: The intraocular pressure remained 21 mmHg or lower for more than 6 months in three of six eyes. Slight bleeding from the iris occurred in one of the 14 procedures, and hypotony (intraocular pressure below 5 mmHg) occurred in one of the 14 procedures. No serious complications developed.Conclusion: Blunt needle revision via the anterior chamber for early failed filtering blebs is a new, simple, and safe procedure.Keywords: glaucoma, trabeculectomy, filtering bleb, needle revision, blunt needl

Relationship between variations in posterior vitreous detachment and visual prognosis in idiopathic epiretinal membranes

Ayumi Ota, Yoshiaki Tanaka, Fumihiko Toyoda, Machiko Shimmura, Nozomi Kinoshita, Hiroko Takano, Akihiro Kakehashi Department of Ophthalmology, Jichi Medical University, Saitama Medical Center, Omiya-ku, Saitama, Japan Purpose: To clarify the relationship between variations in posterior vitreous detachments (PVDs) and visual prognoses in idiopathic epiretinal membranes (ERMs).Methods: In this retrospective, observational, and consecutive case series, we observed variations in PVDs in 37 patients (mean age, 65.7±11.0 years) with ERMs and followed them for 2 years. Three PVD types were found biomicroscopically: no PVD, complete PVD with collapse (C-PVD with collapse), and partial PVD without shrinkage, with persistent vitreous attachment to the macula through the premacular hole of the posterior hyaloid membrane (P-PVD without shrinkage [M]). The best-corrected visual acuity (BCVA) was measured and converted to the logarithm of the minimum angle of resolution (logMAR) BCVA at the first visit and 2 years later.Results: No PVD was observed in 16 of the 37 eyes (mean age, 61.3±11.3 years), C-PVD with collapse in 11 of the 37 eyes (mean age, 69.1±9.9 years), and P-PVD without shrinkage (M) in 10 of the 37 eyes (mean age, 69.3±10.9 years). The logMAR BCVA at the first visit was the worst in the P-PVD without shrinkage (M) group (0.22±0.35) compared with the no-PVD group (−0.019±0.07; P<0.01) and the C-PVD group (0.029±0.08; P<0.05). The logMAR BCVA 2 years later was also worst in the P-PVD without shrinkage (M) group (0.39±0.35) compared with the no-PVD group (0.04±0.13) and the C-PVD with collapse group (0.03±0.09; P<0.05 for both comparisons). The change in the logMAR BCVA over the 2-year follow-up period was worst in the P-PVD without shrinkage (M) group (0.17±0.23) compared with the no-PVD group (0.06±0.14) and the C-PVD with collapse group (0.0009±0.09; P<0.05 for both comparisons).Conclusion: Cases with an ERM with a P-PVD without shrinkage (M) had a worse visual prognosis than those with an ERM with no PVD and C-PVD with collapse. Keywords: posterior vitreous detachment, idiopathic epiretinal membranes, visual prognosi