Misregulation of Scm3p/HJURP Causes Chromosome Instability in Saccharomyces cerevisiae and Human Cells

The kinetochore (centromeric DNA and associated proteins) is a key determinant for high fidelity chromosome transmission. Evolutionarily conserved Scm3p is an essential component of centromeric chromatin and is required for assembly and function of kinetochores in humans, fission yeast, and budding yeast. Overexpression of HJURP, the mammalian homolog of budding yeast Scm3p, has been observed in lung and breast cancers and is associated with poor prognosis; however, the physiological relevance of these observations is not well understood. We overexpressed SCM3 and HJURP in Saccharomyces cerevisiae and HJURP in human cells and defined domains within Scm3p that mediate its chromosome loss phenotype. Our results showed that the overexpression of SCM3 (GALSCM3) or HJURP (GALHJURP) caused chromosome loss in a wild-type yeast strain, and overexpression of HJURP led to mitotic defects in human cells. GALSCM3 resulted in reduced viability in kinetochore mutants, premature separation of sister chromatids, and reduction in Cse4p and histone H4 at centromeres. Overexpression of CSE4 or histone H4 suppressed chromosome loss and restored levels of Cse4p at centromeres in GALSCM3 strains. Using mutant alleles of scm3, we identified a domain in the N-terminus of Scm3p that mediates its interaction with CEN DNA and determined that the chromosome loss phenotype of GALSCM3 is due to centromeric association of Scm3p devoid of Cse4p/H4. Furthermore, we determined that similar to other systems the centromeric association of Scm3p is cell cycle regulated. Our results show that altered stoichiometry of Scm3p/HJURP, Cse4p, and histone H4 lead to defects in chromosome segregation. We conclude that stringent regulation of HJURP and SCM3 expression are critical for genome stability

Quantitative Microscopy Reveals Centromeric Chromatin Stability, Size, and Cell Cycle Mechanisms to Maintain Centromere Homeostasis

The deposited item is a book chapter and is part of the series "Centromeres and Kinetochores" published by the publisher Springer Verlag. The deposited book chapter is a post-print version and has been submitted to peer reviewing. There is no public supplementary material available for this publication. This publication hasn't any creative commons license associated.Centromeres are chromatin domains specified by nucleosomes containing the histone H3 variant, CENP-A. This unique centromeric structure is at the heart of a strong self-templating epigenetic mechanism that renders centromeres heritable. We review how specific quantitative microscopy approaches have contributed to the determination of the copy number, architecture, size, and dynamics of centromeric chromatin and its associated centromere complex and kinetochore. These efforts revealed that the key to long-term centromere maintenance is the slow turnover of CENP-A nucleosomes, a critical size of the chromatin domain and its cell cycle-coupled replication. These features come together to maintain homeostasis of a chromatin locus that directs its own epigenetic inheritance and facilitates the assembly of the mitotic kinetochore.There are no funders and sponsors indicated explicitly in the document.info:eu-repo/semantics/publishedVersio

Photocatalytic treatment of organic pollutants in textile effluent using hydrothermally prepared photocatalytic composite

The hydrothermal synthesis of TiO2 nanoparticles on the surface of calcium aluminosilicate beads of 0.5-1.0 mm in diameter has been carried out under mild hydrothermal conditions. The advantage of immobilisation of the TiO2 nanoparticles by coating on calcium aluminosilicate beads is the ease and efficiency to recover the photocatalyst. The experimental temperature was varied from 150 to 220 degrees C and the experimental duration was 24 h with an autogenous pressure in 1M HCl as solvent. A systematic characterisation of TiO2 coated calcium aluminosilicate beads was carried out by employing analytical techniques like X-ray powder diffraction, SEM, FTIR spectroscopy and positron annihilation lifetime spectroscopy. The photocatalytic treatment of toxic organic pollutants in textile effluents was studied using hydrothermally prepared TiO2 coated calcium aluminosilicate beads. The photodegradation characteristics were studied under both sunlight and ultraviolet light source on the degradation of organic pollutants. Several parameters like concentration and dilution factors of effluents, light source and intensity, initial pH of medium, experimental duration and catalyst loaded into the aqueous medium of textile effluent were studied. The degradation reaction was optimised with respect to the experimental duration and catalytic loaded. The reduction in the chemical oxygen demand (COD) and %T revealed the minimisation of organic pollutants along with colour removal from the textile effluent

Hydrothermal coating of ZnO onto calcium alumino silicate beads and their application in photodegradation of amaranth dye

Hydrothermal coating of ZnO nanoparticles onto the surface of calcium alumino silicate beads was carried out under hydrothermal conditions (T, 220 degrees C; P, 300 psi; duration, 12 h). The reagent grade ZnO and calcium alumino silicate beads (0.5-1.0 mm in diameter, specially prepared as supporting material for ZnO) were used as starting materials along with 1M NaOH as a mineraliser leading to the formation of a new class of photocatalytic material. The effect of the hydrothermal experimental parameters on the coating of ZnO nanoparticles, and the grain morphology, etc. was investigated and thus obtained ZnO coated calcium alumino silicate beads were characterised using X-ray diffraction, SEM, Fourier transform infrared and positron annihilation spectroscopy. Sunlight and ultraviolet light mediated photocatalytic degradation of amaranth dye was studied using hydrothermally prepared ZnO coated calcium alumino silicate beads. The effect of various parameters such as initial dye concentration, catalytic loading, pH of the medium, time duration and light source on the photodegradation of amaranth dye was investigated. Silk industrial effluents containing amaranth dye as a major constituent along with other dyes and dyeing auxiliaries were treated using ZnO coated calcium alumino silicate beads

Photocatalytic degradation of textile effluent using hydrothermally synthesised titania supported molybdenum oxide photocatalyst

The photocatalytic degradation of textile effluent using hydrothermally synthesised titania supported molybdenum oxide photocatalyst at 200 degrees C with an autogenous pressure and experimental duration of 24 h has been reported. A control over the particle size, morphology and crystallinity of the photocatalyst has been studied with respect to the experimental parameters such as nutrient composition, solvent, pH, experimental duration, temperature and pressure. The products synthesised were characterised using XRD, SEM, FTIR, etc. The photodegradation of textile effluent using these composite was investigated under both solar and ultraviolet irradiation. The degradation of textile effluent was checked by the following parameters: chemical oxygen demand (COD), percentage transmission (%T), irradiation time and duration. The preliminary results are highly encouraging and further work is being carried out for the use of these photocatalytic compounds for other organic decomposition

Human centromeric CENP-A chromatin is a homotypic, octameric nucleosome at all cell cycle points

Chromatin assembled with centromere protein A (CENP-A) is the epigenetic mark of centromere identity. Using new reference models, we now identify sites of CENP-A and histone H3.1 binding within the megabase, α-satellite repeat-containing centromeres of 23 human chromosomes. The overwhelming majority (97%) of α-satellite DNA is found to be assembled with histone H3.1-containing nucleosomes with wrapped DNA termini. In both G1 and G2 cell cycle phases, the 2-4% of α-satellite assembled with CENP-A protects DNA lengths centered on 133 bp, consistent with octameric nucleosomes with DNA unwrapping at entry and exit. CENP-A chromatin is shown to contain equimolar amounts of CENP-A and histones H2A, H2B, and H4, with no H3. Solid-state nanopore analyses show it to be nucleosomal in size. Thus, in contrast to models for hemisomes that briefly transition to octameric nucleosomes at specific cell cycle points or heterotypic nucleosomes containing both CENP-A and histone H3, human CENP-A chromatin complexes are octameric nucleosomes with two molecules of CENP-A at all cell cycle phases.BN/Cees Dekker La

Quantitative mapping of fluorescently tagged cellular proteins using FCS-calibrated four-dimensional imaging

The ability to tag a protein at its endogenous locus with a fluorescent protein (FP) enables quantitative understanding of protein dynamics at the physiological level. Genome-editing technology has now made this powerful approach routinely applicable to mammalian cells and many other model systems, thereby opening up the possibility to systematically and quantitatively map the cellular proteome in four dimensions. 3D time-lapse confocal microscopy (4D imaging) is an essential tool for investigating spatial and temporal protein dynamics; however, it lacks the required quantitative power to make the kind of absolute and comparable measurements required for systems analysis. In contrast, fluorescence correlation spectroscopy (FCS) provides quantitative proteomic and biophysical parameters such as protein concentration, hydrodynamic radius, and oligomerization but lacks the capability for high-throughput application in 4D spatial and temporal imaging. Here we present an automated experimental and computational workflow that integrates both methods and delivers quantitative 4D imaging data in high throughput. These data are processed to yield a calibration curve relating the fluorescence intensities (FIs) of image voxels to the absolute protein abundance. The calibration curve allows the conversion of the arbitrary FIs to protein amounts for all voxels of 4D imaging stacks. Using our workflow, users can acquire and analyze hundreds of FCS-calibrated image series to map their proteins of interest in four dimensions. Compared with other protocols, the current protocol does not require additional calibration standards and provides an automated acquisition pipeline for FCS and imaging data. The protocol can be completed in 1 d