Similar Datasets
Project description:The role of DNA sequence in determining replication timing (RT) and chromatin higher order organization remains elusive. To address this question, we have developed an extra-chromosomal replication system (E-BACs) consisting of ?200 kb human bacterial artificial chromosomes (BACs) modified with Epstein-Barr virus (EBV) stable segregation elements. E-BACs were stably maintained as autonomous mini-chromosomes in EBNA1-expressing HeLa or human induced pluripotent stem cells (hiPSCs) and established distinct RT patterns. An E-BAC harboring an early replicating chromosomal region replicated early during S phase, while E-BACs derived from RT transition regions (TTRs) and late replicating regions replicated in mid to late S phase. Analysis of E-BAC interactions with cellular chromatin (4C-seq) revealed that the early replicating E-BAC interacted broadly throughout the genome and preferentially with the early replicating compartment of the nucleus. In contrast, mid- to late-replicating E-BACs interacted with more specific late replicating chromosomal segments, some of which were shared between different E-BACs. Together, we describe a versatile system in which to study the structure and function of chromosomal segments that are stably maintained separately from the influence of cellular chromosome context.
| S-EPMC5829748 | biostudies-literature
Project description:The role of DNA sequence in determining replication timing (RT) and chromatin higher order organization remains elusive. To address this question, we have developed an extra-chromosomal replication system consisting of ~200kb human bacteria artificial chromosomes (BACs) modified with Epstein-Barr virus (EBV) replication origin elements (E-BACs). E-BACs were stably maintained as autonomous mini-chromosomes in both HeLa and human induced pluripotent stem cells (hiPSCs) and established their RT de novo. We applied 4C-seq to evaluate E-BACs' sub-nuclear compartment.
2018-01-29 | GSE102521 | GEO
Project description:The role of DNA sequence in determining replication timing (RT) and chromatin higher order organization remains elusive. To address this question, we have developed an extra-chromosomal replication system consisting of ~200kb human bacteria artificial chromosomes (BACs) modified with Epstein-Barr virus (EBV) replication origin elements (E-BACs). E-BACs were stably maintained as autonomous mini-chromosomes in both HeLa and human induced pluripotent stem cells (hiPSCs) and established their RT de novo. We applied repli-seq to evaluate E-BACs' replication timing.
2018-01-29 | GSE102522 | GEO
Project description:Bacterial artificial chromosomes establish replication timing and sub-nuclear compartment de novo as extra-chromosomal vectors
| PRJNA398470 | ENA
Project description:Bacterial artificial chromosomes establish replication timing and sub-nuclear compartment de novo as extra-chromosomal vectors
| PRJNA429696 | ENA
Project description:BACKGROUND: Human artificial chromosomes (HACs) are potentially useful vectors for gene transfer studies and for functional annotation of the genome because of their suitability for cloning, manipulating and transferring large segments of the genome. However, development of HACs for the transfer of large genomic loci into mammalian cells has been limited by difficulties in manipulating high-molecular weight DNA, as well as by the low overall frequencies of de novo HAC formation. Indeed, to date, only a small number of large (>100 kb) genomic loci have been reported to be successfully packaged into de novo HACs. RESULTS: We have developed novel methodologies to enable efficient assembly of HAC vectors containing any genomic locus of interest. We report here the creation of a novel, bimolecular system based on bacterial artificial chromosomes (BACs) for the construction of HACs incorporating any defined genomic region. We have utilized this vector system to rapidly design, construct and validate multiple de novo HACs containing large (100-200 kb) genomic loci including therapeutically significant genes for human growth hormone (HGH), polycystic kidney disease (PKD1) and beta-globin. We report significant differences in the ability of different genomic loci to support de novo HAC formation, suggesting possible effects of cis-acting genomic elements. Finally, as a proof of principle, we have observed sustained beta-globin gene expression from HACs incorporating the entire 200 kb beta-globin genomic locus for over 90 days in the absence of selection. CONCLUSION: Taken together, these results are significant for the development of HAC vector technology, as they enable high-throughput assembly and functional validation of HACs containing any large genomic locus. We have evaluated the impact of different genomic loci on the frequency of HAC formation and identified segments of genomic DNA that appear to facilitate de novo HAC formation. These genomic loci may be useful for identifying discrete functional elements that may be incorporated into future generations of HAC vectors.
| S-EPMC1182356 | biostudies-literature
Project description:The genome of pathogenic Leptospira interrogans contains two chromosomes. Plasmids and prophages are known to play specific roles in gene transfer in bacteria and can potentially serve as efficient genetic tools in these organisms. Although plasmids and prophage remnants have recently been reported in Leptospira species, their characteristics and potential applications in leptospiral genetic transformation systems have not been fully evaluated.Three extrachromosomal replicons designated lcp1 (65,732 bp), lcp2 (56,757 bp), and lcp3 (54,986 bp) in the L. interrogans serovar Linhai strain 56609 were identified through whole genome sequencing. All three replicons were stable outside of the bacterial chromosomes. Phage particles were observed in the culture supernatant of 56609 after mitomycin C induction, and lcp3, which contained phage-related genes, was considered to be an inducible prophage. L. interrogans-Escherichia coli shuttle vectors, constructed with the predicted replication elements of single rep or rep combined with parAB loci from the three plasmids were shown to successfully transform into both saprophytic and pathogenic Leptospira species, suggesting an essential function for rep genes in supporting auto-replication of the plasmids. Additionally, a wide distribution of homologs of the three rep genes was identified in L. interrogans isolates, and correlation tests showed that the transformability of the shuttle vectors in L. interrogans isolates depended, to certain extent, on genetic compatibility between the rep sequences of both plasmid and host.Three extrachromosomal replicons co-exist in L. interrogans, one of which we consider to be an inducible prophage. The vectors constructed with the rep genes of the three replicons successfully transformed into saprophytic and pathogenic Leptospira species alike, but this was partly dependent on genetic compatibility between the rep sequences of both plasmid and host.
| S-EPMC4338851 | biostudies-literature
Project description:Bacterial artificial chromosome (BAC) mediated transgenesis has proven to be a highly reliable way to obtain accurate transgene expression for in vivo studies of gene expression and function. A rate-limiting step in use of this technology to characterize large numbers of genes has been the process with which BACs can be modified by homologous recombination in Escherichia coli. We report here a highly efficient method for modifying BACs by using a novel set of shuttle vectors that contain the R6Kgamma origin for DNA replication, the E. coli RecA gene for recombination, and the SacB gene for negative selection. These new vectors greatly increased the ease with which one can clone the shuttle vectors, as well as screen for co-integrated and resolved clones. Furthermore, we simplify the shuttle vector cloning to one step by incorporation of a "built-in" resolution cassette for rapid removal of the unwanted vector sequences. This new system has been used to modify a dozen BACs. It is well suited for efficient production of modified BACs for use in a variety of in vivo studies.
| S-EPMC187570 | biostudies-literature
Project description:Aneuploidy is a hallmark of cancer and underlies genetic disorders characterized by severe developmental defects, yet the molecular mechanisms explaining its effects on cellular physiology remain elusive. Here we show, using a series of human cells with defined aneuploid karyotypes, that gain of a single chromosome increases genomic instability. Next-generation sequencing and SNP-array analysis reveal accumulation of chromosomal rearrangements in aneuploids, with break point junction patterns suggestive of replication defects. Trisomic and tetrasomic cells also show increased DNA damage and sensitivity to replication stress. Strikingly, we find that aneuploidy-induced genomic instability can be explained by the reduced expression of the replicative helicase MCM2-7. Accordingly, restoring near-wild-type levels of chromatin-bound MCM helicase partly rescues the genomic instability phenotypes. Thus, gain of chromosomes triggers replication stress, thereby promoting genomic instability and possibly contributing to tumorigenesis.
| S-EPMC4756715 | biostudies-literature
Project description:De novo assembled alphoid(tetO)-type human artificial chromosomes (HACs) represent a novel promising generation of high capacity episomal vectors. Their function and persistence, and any adverse effects, in various cell types in live animals, have not, however, been explored. In this study we transferred the alphoid(tetO)-HAC into mouse ES cells and assessed whether the presence of this extra chromosome affects their pluripotent properties. Alphoid(tetO)-HAC-bearing ES cells were indistinguishable from their wild-type counterparts: they retained self-renewal potential and full capacity for multilineage differentiation during mouse development, whereas the HAC itself was mitotically and transcriptionally stable during this process. Our data provide the first example of fully synthetic DNA behaving like a normal chromosome in cells of living animals. It also opens a new perspective into functional genetic studies in laboratory animals as well as stem cell-based regenerative medicine.
| S-EPMC4614155 | biostudies-literature