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Here, we report the successful design, construction, and characterization of a 770-kilobase synthetic yeast chromosome II (synII). Our study incorporates characterization at multiple levels—including phenomics, transcriptomics, proteomics, chromosome segregation, and replication analysis—to provide a thorough and comprehensive analysis of a synthetic chromosome. Our Trans-Omics analyses reveal a modest but potentially relevant pervasive up-regulation of translational machinery observed in synII, mainly caused by the deletion of 13 transfer RNAs. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by loxP -mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium, which is related to misregulation of the high-osmolarity glycerol response. Despite the subtle differences, the synII strain shows highly consistent biological processes comparable to the native strain.

DNA topoisomerases are thought to play a critical role in transcription, replication and recombination as well as in the condensation and segregation of sister duplexes during cell division. Here, we used high-resolution two-dimensional agarose gel electrophoresis to study the replication intermediates and final products of small circular and linear minichromosomes of Saccharomyces cerevisiae in the presence and absence of DNA topoisomerase 2. The results obtained confirmed that whereas for circular minichromosomes, catenated sister duplexes accumulated in the absence of topoisomerase 2, linear YACs were able to replicate and segregate regardless of this topoisomerase. The patterns of replication intermediates for circular and linear YACs displayed significant differences suggesting that DNA supercoiling might play a key role in the modulation of replication fork progression. Altogether, this data supports the notion that for linear chromosomes the torsional tension generated by transcription and replication dissipates freely throughout the telomeres.

Key Points Chromosomal DNA is packaged with histones into nucleosomes. Following replication, sister-chromatid segregation is orchestrated to ensure that each daughter cell receives a complete set of chromosomes. The centromere is the chromosomal region responsible for formation of the kinetochore, which mediates attachment to spindle microtubules and chromosome movement. In organisms with complex centromeres the site of kinetochore assembly is epigenetically determined and is not strictly governed by primary DNA sequence. The key determining factor in specifying the site of kinetochore assembly is the deposition of the centromere-specific histone H3 variant CENP-A. In tissue-culture cells, new CENP-A is deposited during late mitosis and through G1, independently of DNA replication. A portion of the histone fold domain of CENP-A is necessary and sufficient for targeting to centromeres. CENP-A nucleosomes depleted during S phase replication are restored later in the cell cycle by de novo deposition into 'gaps' or by replacement of H3 nucleosomes. Numerous factors have been identified that are required for CENP-A localization, but the chromatin assembly proteins that are directly required for propagation of CENP-A chromatin are not known. Non-coding transcripts and transcription factors are associated with centromeres, and transcription might be linked to CENP-A chromatin assembly in some systems. CENP-B binding sites and flanking centric heterochromatin influence the establishment of CENP-A chromatin on naive templates. The correct location and structure of centromeric chromatin is essential for accurate chromosome segregation. This Review brings together recent findings relating to the centromeric histone H3 variant, CENP-A, and discusses possible models for the establishment and propagation of centromeric chromatin. The assembly of just a single kinetochore at the centromere of each sister chromatid is essential for accurate chromosome segregation during cell division. Surprisingly, despite their vital function, centromeres show considerable plasticity with respect to their chromosomal locations and activity. The establishment and maintenance of centromeric chromatin, and therefore the location of kinetochores, is epigenetically regulated. The histone H3 variant CENP-A is the key determinant of centromere identity and kinetochore assembly. Recent studies have identified many factors that affect CENP-A localization, but their precise roles in this process are unknown. We build on these advances and on new information about the timing of CENP-A assembly during the cell cycle to propose new models for how centromeric chromatin is established and propagated.

In a mouse model of Huntington's disease, synaptic activation of NMDA receptors induces the formation of huntingtin-containing inclusions, rendering neurons more resistant to death in vivo and in vitro . In contrast, stimulation of extrasynaptic NMDA receptors increases neuronal vulnerability by preventing inclusion formation. Huntington's disease is caused by an expanded CAG repeat in the gene encoding huntingtin ( HTT ), resulting in loss of striatal and cortical neurons. Given that the gene product is widely expressed, it remains unclear why neurons are selectively targeted. Here we show the relationship between synaptic and extrasynaptic activity, inclusion formation of mutant huntingtin protein (mtHtt) and neuronal survival. Synaptic N -methyl- D -aspartate–type glutamate receptor (NMDAR) activity induces mtHtt inclusions via a T complex-1 (TCP-1) ring complex (TRiC)-dependent mechanism, rendering neurons more resistant to mtHtt-mediated cell death. In contrast, stimulation of extrasynaptic NMDARs increases the vulnerability of mtHtt-containing neurons to cell death by impairing the neuroprotective cyclic AMP response element–binding protein (CREB)–peroxisome proliferator–activated receptor-γ coactivator-1α (PGC-1α) cascade and increasing the level of the small guanine nucleotide–binding protein Rhes, which is known to sumoylate and disaggregate mtHtt. Treatment of transgenic mice expressing a yeast artificial chromosome containing 128 CAG repeats (YAC128) with low-dose memantine blocks extrasynaptic (but not synaptic) NMDARs and ameliorates neuropathological and behavioral manifestations. By contrast, high-dose memantine, which blocks both extrasynaptic and synaptic NMDAR activity, decreases neuronal inclusions and worsens these outcomes. Our findings offer a rational therapeutic approach for protecting susceptible neurons in Huntington's disease.

Faithful chromosome segregation is essential for the maintenance of genomic integrity and requires functional centromeres. Centromeres are epigenetically defined by the histone H3 variant, centromere protein A (CENP-A). Here we highlight current knowledge regarding CENP-A-containing chromatin structure, specification of centromere identity, regulation of CENP-A deposition and possible contribution to cancer formation and/or progression. CENP-A overexpression is common among many cancers and predicts poor prognosis. Overexpression of CENP-A increases rates of CENP-A deposition ectopically at sites of high histone turnover, occluding CCCTC-binding factor (CTCF) binding. Ectopic CENP-A deposition leads to mitotic defects, centromere dysfunction and chromosomal instability (CIN), a hallmark of cancer. CENP-A overexpression is often accompanied by overexpression of its chaperone Holliday Junction Recognition Protein (HJURP), leading to epigenetic addiction in which increased levels of HJURP and CENP-A become necessary to support rapidly dividing p53 deficient cancer cells. Alterations in CENP-A posttranslational modifications are also linked to chromosome segregation errors and CIN. Collectively, CENP-A is pivotal to genomic stability through centromere maintenance, perturbation of which can lead to tumorigenesis.

Chromosome instability is a key component of cancer progression and many heritable diseases. Understanding why some chromosomes are more unstable than others could provide insight into understanding genome integrity. Here we systematically investigate the spontaneous chromosome loss for all sixteen chromosomes in Saccharomyces cerevisiae in order to elucidate the mechanisms underlying chromosome instability. We observed that the stability of different chromosomes varied more than 100-fold. Consistent with previous studies on artificial chromosomes, chromosome loss frequency was negatively correlated to chromosome length in S. cerevisiae diploids, triploids and S. cerevisiae-S. bayanus hybrids. Chromosome III, an equivalent of sex chromosomes in budding yeast, was found to be the most unstable chromosome among all cases examined. Moreover, similar instability was observed in chromosome III of S. bayanus, a species that diverged from S. cerevisiae about 20 million years ago, suggesting that the instability is caused by a conserved mechanism. Chromosome III was found to have a highly relaxed spindle checkpoint response in the genome. Using a plasmid stability assay, we found that differences in the centromeric sequence may explain certain aspects of chromosome instability. Our results reveal that even under normal conditions, individual chromosomes in a genome are subject to different levels of pressure in chromosome loss (or gain).

Human artificial chromosome (HAC)-based vectors offer a promising system for delivery and expression of full-length human genes of any size. HACs avoid the limited cloning capacity, lack of copy number control, and insertional mutagenesis caused by integration into host chromosomes that plague viral vectors. We previously described a synthetic HAC that can be easily eliminated from cell populations by inactivation of its conditional kinetochore. Here, we demonstrate the utility of this HAC, which has a unique gene acceptor site, for delivery of full-length genes and correction of genetic deficiencies in human cells. A battery of functional tests was performed to demonstrate expression of NBS1 and VHL genes from the HAC at physiological levels. We also show that phenotypes arising from stable gene expression can be reversed when cells are \"cured\" of the HAC by inactivating its kinetochore in proliferating cell populations, a feature that provides a control for phenotypic changes attributed to expression of HAC-encoded genes. This generation of human artificial chromosomes should be suitable for studies of gene function and therapeutic applications.

Key Points Genetic engineering has traditionally been done in Escherichia coli using restriction enzymes to cleave DNA, and using DNA ligases to join them. However, there are several limitations to this approach, especially when large DNA molecules require engineering, because even rare restriction enzymes occur over large stretches of DNA. The generation of transgenic and mouse knockout constructs in E. coli is also hampered by the difficulty of finding appropriately placed restriction-enzyme cleavage sites. Genetic engineering in yeast alleviates these problems because it relies on homologous recombination rather than on restriction enzymes and DNA ligases to generate recombinant DNA molecules. A principal limitation of genetic engineering in yeast is that yeast artificial cloning vectors (YACs), developed for cloning large DNA molecules, are often unstable, and YAC transgenic mice are difficult to make. As a result, more stable vectors, such as bacterial artificial chromosomes (BACs), are often used instead. Phage-based E. coli recombination systems have been developed that now allow large DNA molecules cloned into BACs to be modified by homologous recombination, similarly to what occurs in yeast. These E. coli recombination systems have many of the advantages of yeast recombination, but few of its disadvantages. This new form of chromosome engineering, termed recombineering, makes it possible to introduce virtually any type of mutation into a BAC using PCR-amplified, linear, double-stranded DNA targeting cassettes that have short regions of homology at their ends, or single-stranded oligonucleotides. Recombineering greatly decreases the time it takes to create transgenic mouse models by conventional means. It also facilitates many kinds of genomic experiment that have otherwise been difficult to carry out, and it should enhance functional genomic studies by providing better mouse models and a more refined genetic analysis of the mouse genome. Highly efficient phage-based Escherichia coli homologous recombination systems have recently been developed that enable genomic DNA in bacterial artificial chromosomes to be modified and subcloned, without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombinogenic engineering or recombineering, is efficient and greatly decreases the time it takes to create transgenic mouse models by traditional means. Recombineering also facilitates many kinds of genomic experiment that have otherwise been difficult to carry out, and should enhance functional genomic studies by providing better mouse models and a more refined genetic analysis of the mouse genome.

Hutchinson-Gilford progeria syndrome (HGPS), a devastating premature aging disease, is caused by a point mutation in the lamin A gene (LMNA). This mutation constitutively activates a cryptic splice donor site, resulting in a mutant lamin A protein known as progerin. Recent studies have demonstrated that progerin is also produced at low levels in normal human cells and tissues. However, the cause-and-effect relationship between normal aging and progerin production in normal individuals has not yet been determined. In this study, we have shown in normal human fibroblasts that progressive telomere damage during cellular senescence plays a causative role in activating progerin production. Progressive telomere damage was also found to lead to extensive changes in alternative splicing in multiple other genes. Interestingly, elevated progerin production was not seen during cellular senescence that does not entail telomere shortening. Taken together, our results suggest a synergistic relationship between telomere dysfunction and progerin production during the induction of cell senescence, providing mechanistic insight into how progerin may participate in the normal aging process.

We report the design, synthesis, and assembly of the 1.08-mega-base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including \"watermark\" sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication.