Synthesis, debugging and effects of synthetic chromosome consolidation: synVI and beyond
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Saccharomyces Cerevisiae (ncbitaxon:4932)
SUBMITTER: 
Jef D. Boeke
PROVIDER: MSV000080584 | MassIVE | Tue Feb 28 07:33:00 GMT 2017
REPOSITORIES: MassIVE
Similar Datasets
Project description:Background. Most methods for constructing aneuploid yeast strains that have gained a specific chromosome rely on spontaneous failures of cell division fidelity. In Saccharomyces cerevisiae, extra chromosomes can be obtained when errors in meiosis or mitosis lead to nondisjunction, or when nuclear breakdown occurs in heterokaryons. We describe a strategy for constructing N+1 disomes that does not require such spontaneous failures. The method combines two well-characterized genetic tools: a conditional centromere that transiently blocks disjunction of one specific chromosome, and a duplication marker assay that identifies disomes among daughter cells. To test the strategy, we targeted chromosomes III, IV, and VI for duplication. Results. The centromere of each target chromosome was replaced by a conditional centromere that can be blocked by growth in galactose, and ura3::HIS3, a duplication marker. Transient exposure to galactose induced the appearance of colonies carrying duplicated markers for chromosomes III or IV, but not VI. Microarray-based comparative genomic hybridization (CGH) confirmed that disomic strains carrying extra chromosome III or IV were generated. Chromosome VI contains several genes known to be deleterious when overexpressed, including the beta-tubulin gene TUB2. To test whether a tubulin stoichiometry imbalance prevents viability in cells carrying an extra chromosome VI, we supplied the parent strain with extra copies of the alpha-tubulin gene TUB1, then induced nondisjunction. Galactose-dependent chromosome VI disomes were produced, as revealed by CGH. Some chromosome VI disomes also carried extra, unselected copies of additional chromosomes. Conclusions. This method causes efficient nondisjunction of a targeted chromosome and allows resulting disomic cells to be identified and maintained. We used the method to test the role of tubulin imbalance in the apparent lethality of disomic chromosome VI. Our results indicate that a tubulin imbalance is necessary for disomic VI lethality, but it may not be the only dosage-dependent effect. Keywords: comparative genomic hybridization, CGH
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Project description:Eukaryotic cell proliferation requires chromosome replication and precise segregation to ensure daughter cells have identical genomic copies. The genus Plasmodium, the causative agent of malaria, has developed remarkable characteristic of nuclear division throughout its lifecycle to meet some peculiar and unique challenges of DNA replication and chromosome segregation. The parasite undergoes atypical endomitosis and endoreduplication with an intact nuclear membrane and intranuclear mitotic spindle. To understand diverse modes of cell division in Plasmodium we have studied the behaviour and composition of a key part of the mitotic apparatus, the outer kinetochore Ndc80 complex that attaches the centromere of chromosomes to the microtubules of the mitotic spindle. Using Ndc80 live-cell imaging in Plasmodium berghei we observe dynamic changes throughout proliferative life-stages including highly unusual kinetochore arrangements in sexual stages of the parasite. Furthermore, we identify a divergent Spc24 component of Ndc80 complex, previously thought to be missing, and completing a canonical, albeit unusual, Ndc80 complex structure. Altogether our studies reveal the kinetochore as an ideal starting point towards understanding non-canonical modes of chromosome segregation and cell division in Plasmodium.
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Project description:A chromosome size-dependent bias in meiotic recombination is in place to ensure that homologous chromosome pairing occurs for all chromosomes, including the smallest ones. This bias is clearly detectable during the assembly of the meiotic protein axis, the structure that compacts meiotic chromosomes and promotes recombination.To investigate the origin of the size bias, we mapped genome-wide occupancy of the meiotic axis protein Red1 in yeast strains containing chromosome fusions and synthetic chromosomes. Meiosis studies of the fusion and synthetic chromosomes further revealed that core centromeres influence the deposition of the axial element protein Red1 over distances >100kb, while pericentromeric regions co-evolved to reduce Red1 binding near centromeres and spread out Red1 along the chromosomes.
2021-05-21 | GSE114731 | GEO
Project description:We designed and synthesized synI, which is ~21.4% shorter than native chrI, the smallest chromosome in Saccharomyces cerevisiae. SynI was designed for attachment to another synthetic chromosome due to concerns surrounding potential instability and karyotype imbalance, and is now attached to synIII, yielding the first synthetic yeast fusion chromosome. We constructed additional fusion chromosomes to investigate effects of fusions on nuclear function. We observed unexpected loops and twisted structures in chrIII-I and chrIX-III-I fusion chromosomes dependent on silencing protein Sir3. ChrI faces special challenges in assuring meiotic crossovers required for efficient homolog disjunction. Centromere deletions engineered into fusion chromosomes revealed opposing effects of core centromeres and pericentromeres in modulating deposition of meiotic recombination protein Red1. These effects extended over >100kb, to disproportionally promote meiotic recombination of small chromosomes like chrI. These findings reveal the power of synthetic genomics to uncover new biology and deconvolute complex biological systems.
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Project description:Background. Most methods for constructing aneuploid yeast strains that have gained a specific chromosome rely on spontaneous failures of cell division fidelity. In Saccharomyces cerevisiae, extra chromosomes can be obtained when errors in meiosis or mitosis lead to nondisjunction, or when nuclear breakdown occurs in heterokaryons. We describe a strategy for constructing N+1 disomes that does not require such spontaneous failures. The method combines two well-characterized genetic tools: a conditional centromere that transiently blocks disjunction of one specific chromosome, and a duplication marker assay that identifies disomes among daughter cells. To test the strategy, we targeted chromosomes III, IV, and VI for duplication. Results. The centromere of each target chromosome was replaced by a conditional centromere that can be blocked by growth in galactose, and ura3::HIS3, a duplication marker. Transient exposure to galactose induced the appearance of colonies carrying duplicated markers for chromosomes III or IV, but not VI. Microarray-based comparative genomic hybridization (CGH) confirmed that disomic strains carrying extra chromosome III or IV were generated. Chromosome VI contains several genes known to be deleterious when overexpressed, including the beta-tubulin gene TUB2. To test whether a tubulin stoichiometry imbalance prevents viability in cells carrying an extra chromosome VI, we supplied the parent strain with extra copies of the alpha-tubulin gene TUB1, then induced nondisjunction. Galactose-dependent chromosome VI disomes were produced, as revealed by CGH. Some chromosome VI disomes also carried extra, unselected copies of additional chromosomes. Conclusions. This method causes efficient nondisjunction of a targeted chromosome and allows resulting disomic cells to be identified and maintained. We used the method to test the role of tubulin imbalance in the apparent lethality of disomic chromosome VI. Our results indicate that a tubulin imbalance is necessary for disomic VI lethality, but it may not be the only dosage-dependent effect. Keywords: comparative genomic hybridization, CGH Candidate disomes (Ura+His+) were compared to their euploid parents by CGH. 27 candidate strains were examined with 27 arrays. 4 candidates targeted chromosome III (3 induced, 1 spontaneous), 14 candidates targeted chromosome IV (8 induced, 6 spontaneous), and 9 candidates targeted chromosome VI (all induced).
2009-01-13 | E-GEOD-14377 | biostudies-arrayexpress
Project description:The Sc2.0 project is building a eukaryotic synthetic genome from scratch. A major milestone has been achieved with all individual Sc2.0 chromosomes assembled. Here, we describe consolidation of multiple synthetic chromosomes using advanced endoreduplication intercrossing with tRNA expression cassettes to generate a strain with 6.5 synthetic chromosomes. The 3D chromosome organization and transcript isoform profiles were evaluated using Hi-C and long-read direct RNA sequencing. We developed CRISPR Directed Biallelic URA3-assisted Genome Scan, or “CRISPR D-BUGS”, to map phenotypic variants caused by specific designer modifications, known as “bugs”. We first fine-mapped a bug in synthetic chromosome II (synII), and then discovered a combinatorial interaction associated with synIII and synX, revealing an unexpected genetic interaction that links transcriptional regulation, inositol metabolism and tRNASerCGA abundance. Finally, to expedite consolidation, we employed chromosome substitution to incorporate the largest chromosome (synIV), thereby consolidating >50% of the Sc2.0 genome in one strain.
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Project description:Herein we report the successful design, construction and characterization of a 770 kb synthetic yeast chromosome II (synII). Our study incorporates characterization at multiple levels, including phenomics, transcriptomics, proteomics, chromosome segregation and replication analysis to provide the most thorough and comprehensive analysis of a synthetic chromosome. Our “Trans-Omics” analyses reveal a modest but potentially significant pervasive up-regulation of translational machinery observed in synII is mainly caused by the deletion of 13 tRNAs. By both complementation assays and SCRaMbLE, we targeted and debuged the origin of a growth defect at 37°C in glycerol medium, which is related to misregulation of the HOG response. Despite the subtle differences, the synII strain shows highly consistent biological processes comparable to the native strain.
2018-04-19 | GSE89611 | GEO