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Summary Recent studies have revealed the importance of Ki-67 and the chromosome periphery in chromosome structure and segregation, but little is known about this elusive chromosome compartment. Here we used correlative light and serial block-face scanning electron microscopy, which we term 3D-CLEM, to model the entire mitotic chromosome complement at ultra-structural resolution. Prophase chromosomes exhibit a highly irregular surface appearance with a volume smaller than metaphase chromosomes. This may be because of the absence of the periphery, which associates with chromosomes only after nucleolar disassembly later in prophase. Indeed, the nucleolar volume almost entirely accounts for the extra volume found in metaphase chromosomes. Analysis of wild-type and Ki-67-depleted chromosomes reveals that the periphery comprises 30%–47% of the entire chromosome volume and more than 33% of the protein mass of isolated mitotic chromosomes determined by quantitative proteomics. Thus, chromatin makes up a surprisingly small percentage of the total mass of metaphase chromosomes., Graphical Abstract, Highlights • 3D-CLEM combines light and serial block-face scanning electron microscopy • The complete architecture of all 46 human chromosomes has been defined • A large portion of mitotic chromosomes is not composed of chromatin • Chromosome volumes determined by light and electron microscopy differ dramatically, Booth et al. develop a microscopy method called 3D-CLEM. Using this approach, they find that a large portion of mitotic chromosomes is not composed of chromatin, challenging classical perceptions of chromosomes.

It is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity. However, the pathways leading to the formation and maintenance of centromere chromatin remain poorly characterized due to difficulties of analysis of centromeric repeats in native chromosomes. To address this problem, in our previous studies we generated a human artificial chromosome (HAC) whose centromere contains a synthetic alpha-satellite (alphoid) DNA array containing the tetracycline operator, the alphoidtetO-HAC. The presence of tetO sequences allows the specific targeting of the centromeric region in the HAC with different chromatin modifiers fused to the tetracycline repressor. The alphoidtetO-HAC has been extensively used to investigate protein interactions within the kinetochore and to define the epigenetic signature of centromeric chromatin to maintain a functional kinetochore. In this study, we developed a novel synthetic HAC containing two alphoid DNA arrays with different targeting sequences, tetO, lacO and gal4, the alphoidhybrid-HAC. This new HAC can be used for detailed epigenetic engineering studies because its kinetochore can be simultaneously or independently targeted by different chromatin modifiers and other fusion proteins.

A series of (Co-Ti-O)/Bi-Ti-O multilayer films with 1-65 Co-Ti-O magnetic layers (MLs) are prepared by tandem sputtering method. The effects of the multilayers on the electromagnetic responses were investigated in the frequency range from 0.1 to 10 GHz. The multilayer films demonstrate soft-magnetic performance, maintain low loss of permeability (mu) up to a few GHz, and exhibit a clear peak of mu '' at their resonance frequency (f(r)). f(r) increase from 1.9 to 4.7 GHz with increasing MLs from 1 to 65. The multilayer films show a notable indentation of susceptance (B) at approximately f(r), which is not observed in a Bi-Ti-O monolayer film. (C) 2013 The Japan Society of Applied Physics

Human artificial chromosomes (HACs), which carry a fully functional centromere and are maintained as a single-copy episome, are not associated with random mutagenesis and offer greater control over expression of ectopic genes on the HAC. Recently, we generated a HAC with a conditional centromere, which includes the tetracycline operator (tet-O) sequence embedded in the alphoid DNA array. This conditional centromere can be inactivated, loss of the alphoid(tet-O) (tet-O HAC) by expression of tet-repressor fusion proteins. In this report, we describe adaptation of the tet-O HAC vector for gene delivery and gene expression in human cells. A loxP cassette was inserted into the tet-O HAC by homologous recombination in chicken DT40 cells following a microcell-mediated chromosome transfer (MMCT). The tet-O HAC with the loxP cassette was then transferred into Chinese hamster ovary cells, and EGFP transgene was efficiently and accurately incorporated into the tet-O HAC vector. The EGFP transgene was stably expressed in human cells after transfer via MMCT. Because the transgenes inserted on the tet-O HAC can be eliminated from cells by HAC loss due to centromere inactivation, this HAC vector system provides important novel features and has potential applications for gene expression studies and gene therapy.