Sequencing Micronuclei Reveals the Landscape of Chromosomal Instability

Genome instability (GIN) is a main contributing factor to congenital and somatic diseases, but its sporadic occurrence in individual cell cycles makes it difficult to study mechanistically. One profound manifestation of GIN is the formation of micronuclei (MN), the engulfment of chromosomes or chromosome fragments in their own nuclear structures separate from the main nucleus. Here, we developed MN-seq, an approach for sequencing the DNA contained within micronuclei. We applied MN-seq to mice with mutations in Mcm4 and Rad9a, which disrupt DNA replication, repair, and damage responses. Data analysis and simulations show that centromere presence, fragment length, and a heterogenous landscape of chromosomal fragility all contribute to the patterns of DNA present within MN. In particular, we show that long genes, but also gene-poor regions, are associated with chromosome breaks that lead to the enrichment of particular genomic sequences in MN, in a genetic background-specific manner. Finally, we introduce single-cell micronucleus sequencing (scMN-seq), an approach to sequence the DNA present in MN of individual cells. Together, sequencing micronuclei provides a systematic approach for studying GIN and reveals novel molecular associations with chromosome breakage and segregation.

De novo centromere formation on chromosome fragments with an inactive centromere in maize (Zea mays)

The B chromosome of maize undergoes nondisjunction at the second pollen mitosis as part of its accumulation mechanism. Previous work identified 9-Bic-1 (9-B inactivated centromere-1), which comprises an epigenetically silenced B chromosome centromere that was translocated to the short arm of chromosome 9(9S). This chromosome is stable in isolation, but when normal B chromosomes are added to the genotype, it will attempt to undergo nondisjunction during the second pollen mitosis and usually fractures the chromosome in 9S. These broken chromosomes allow a test of whether the inactive centromere is reactivated or whether a de novo centromere is formed elsewhere on the chromosome to allow recovery of fragments. Breakpoint determination on the B chromosome and chromosome 9 showed that mini chromosome B1104 has the same breakpoint as 9-Bic-1 in the B centromere region and includes a portion of 9S. CENH3 binding was found on the B centromere region and on 9S, suggesting both centromere reactivation and de novo centromere formation. Another mini chromosome, B496, showed evidence of rearrangement, but it also only showed evidence for a de novo centromere. Other mini chromosome fragments recovered were directly derived from the B chromosome with breakpoints concentrated near the centromeric knob region, which suggests that the B chromosome is broken at a low frequency due to the failure of the sister chromatids to separate at the second pollen mitosis. Our results indicate that both reactivation and de novo centromere formation could occur on fragments derived from the progenitor possessing an inactive centromere.

Evaluation of extracts of wild Cannabis sativa L. for genotoxicity and phytochemical composition

Cannabis sativa L. is used as medicine and narcotic in Lesotho. Phytochemical composition and total phenolics content (TPC) for hexane, chloroform, ethyl acetate and methanol extracts of aerial parts of C. sativa were determined. Ethyl acetate extract (0.1875, 0.375 and 0.75 mg mL-1) and methanol extract (0.75, 1.5 and 3.0 mg mL-1) were evaluated for cytotoxicity, genotoxicity and modulation of cyclophosphamide (CP, 1.25 mg mL-1)- and ethylmethane sulphonate (EMS, 0.25 mg mL-1)-induced genotoxicity using Allium cepa root meristem assay. CP or EMS did not reduce mitotic index (MI) of cells, hence not cytotoxic when compared with negative control using the t-test (p>0.05), but genotoxic. Both extracts were genotoxic with methanol extract also being cytotoxic. Genotoxicity was the number of aberrant cells per 100 mitotic cells. Modulatory effect (ME) was obtained by comparing mutagen-induced genotoxicity with mixture-induced genotoxicity and expressed as the number of units of mutagen-induced genotoxicity that equalled the mixture-induced genotoxicity. ME was either positive or negative and significant only if ME = ≥ 2. Both extracts were genotoxic with methanol extract also being cytotoxic. Aberrations observed were sticky chromosomes, c-metaphase, anaphase and telophase bridges, chromosome fragments and laggards. Mixture of methanol extract with CP or EMS was more genotoxic (+ME range = 1.61-11.89) than the mutagen or extract alone which suggested synergistic interaction. Mixture of ethyl acetate extract with CP induced insignificant +ME. Mixture of ethylacetate extract with EMS was significantly more genotoxic (+ME = 2.20) than EMS only at high extract concentration. The methanol and ethylacetate extracts of C. sativa were not anti-genotoxic to CP- or EMS- induced genotoxicity. TPCs for hexane, chloroform, ethyl acetate and methanol extracts were 39831.46, 2544.94, 2438.20 and 56601.12 mg GAE/gram dry weight respectively. The differences in the cytotoxicity and MEs of the extracts were attributed to differences in phytochemical composition of extracts.

Identification of QTL related to anther color and hull color by RAD sequencing in a RIL population of Setaria italica

Background Foxtail millet (Setaria italica) is one of the oldest domesticated crops and has been considered as an ideal model plant for C4 grasses. It has abundant type of anther and hull colors which is not only a most intuitive morphological marker for color selection in seed production, but also has very important biological significance for the study of molecular mechanism of regulating the synthesis and metabolism of flavonoids and lignin. However, only a few genetic studies have been reported for anther color and hull color in foxtail millet. Results Quantitative trait loci (QTL) analysis for anther color and hull color was conducted using 400 F6 and F7 recombinant inbreed lines (RILs) derived from a cross between parents Yugu18 and Jigu19. Using restriction-site associated DNA sequencing, 43,001 single-nucleotide polymorphisms (SNPs) and 3,022 indels were identified between both the parents and the RILs. A total of 1,304 bin markers developed from the SNPs and indels were used to construct a genetic map that spanned 2196 cM of the foxtail millet genome with an average of 1.68 cM/bin. Combined with this genetic map and the phenotypic data observed in two locations for two years, two QTL located on chromosome 6 (Chr6) in a 1.215-Mb interval (33,627,819–34,877,940 bp) for anther color (yellow - white) and three QTL located on Chr1 in a 6.23-Mb interval (1–6,229,734 bp) for hull color (gold-reddish brown) were detected. To narrow the QTL regions identified from the genetic map and QTL analysis, we developed a new method named “inconsistent rate analysis” and efficiently narrowed the QTL regions of anther color into a 60-kb interval (34.13–34.19 Mb) in Chr6, and narrowed the QTL regions of hull color into 70-kb (5.43–5.50 Mb) and 30-kb (5.69–5.72 Mb) intervals in Chr1. Two genes (Seita.6G228600.v2.2 and Seita.6G228700.v2.2) and a cinnamyl alcohol dehydrogenase (CAD) gene (Seita.1G057300.v2.2) with amino acid changes between the parents detected by whole-genome resequencing were identified as candidate genes for anther and hull color, respectively. Conclusions This work presents the related QTL and candidate genes of anther and hull color in foxtail millet and developed a new method named inconsistent rate analysis to detect the chromosome fragments linked with the quality trait in RILs. This is the first study of the QTL related to hull color in foxtail millet and clarifying that the CAD gene (Seita.1G057300.v2.2) is the key gene responsible for this trait. It lays the foundation for further cloning of the functional genes and provides a powerful tool to detect the chromosome fragments linked with quality traits in RILs.

Kinetochore-independent mechanisms of sister chromosome separation

Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore- independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, we discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, we screened 117 RNAi gene knockdowns for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on our analysis of EB1 localization and knockdown phenotypes, we propose that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation.

Sequencing Red Fox Y Chromosome Fragments to Develop Phylogenetically Informative SNP Markers and Glimpse Male-Specific Trans-Pacific Phylogeography

The red fox (Vulpes vulpes) has a wide global distribution with many ecotypes and has been bred in captivity for various traits, making it a useful evolutionary model system. The Y chromosome represents one of the most informative markers of phylogeography, yet it has not been well-studied in the red fox due to a lack of the necessary genomic resources. We used a target capture approach to sequence a portion of the red fox Y chromosome in a geographically diverse red fox sample, along with other canid species, to develop single nucleotide polymorphism (SNP) markers, 13 of which we validated for use in subsequent studies. Phylogenetic analyses of the Y chromosome sequences, including calibration to outgroups, confirmed previous estimates of the timing of two intercontinental exchanges of red foxes, the initial colonization of North America from Eurasia approximately half a million years ago and a subsequent continental exchange before the last Pleistocene glaciation (~100,000 years ago). However, in contrast to mtDNA, which showed unidirectional transfer from Eurasia to North America prior to the last glaciation, the Y chromosome appears to have been transferred from North America to Eurasia during this period. Additional sampling is needed to confirm this pattern and to further clarify red fox Y chromosome phylogeography.

Cytokinesis-Block Micronucleus Cytome Assay Evolution into a More Comprehensive Method to Measure Chromosomal Instability

This review describes the cytokinesis-block micronucleus (CBMN) cytome assay and its evolution into a molecular cytogenetic method of chromosomal instability (CIN). Micronuclei (MNi) originate from whole chromosomes or chromosome fragments that fail to segregate to the poles of the cell during mitosis. These lagging chromosomes are excluded from the daughter nuclei and are enveloped in their own membrane to form MNi. The CBMN assay was developed to allow MNi to be scored exclusively in once-divided binucleated cells, which enables accurate measurement of chromosome breakage or loss without confounding by non-dividing cells that cannot express MNi. The CBMN assay can be applied to cell lines in vitro and cells such as lymphocytes that can be stimulated to divide ex vivo. In the CBMN assay, other CIN biomarkers such as nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) are also measured. Use of centromere, telomere, and chromosome painting probes provides further insights into the mechanisms through which MNi, NPBs and NBUDs originate. Measurement of MNi is also important because entrapment within a micronucleus may cause chromosomes to shatter and, after nuclear reintegration, become rearranged. Additionally, leakage of DNA from MNi can stimulate inflammation via the cyclic GMP-AMP Synthase—Stimulator of Interferon Genes (cGAS-STING) DNA sensing mechanism of the innate immune system.

Kinetochore-independent mechanisms of sister chromosome separation

ABSTRACTAlthough kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore-independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, we discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, we screened 117 RNAi gene knockdowns for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on our analysis of EB1 localization and knockdown phenotypes, we propose that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation.AUTHOR SUMMARYKinetochores, the site on the chromosomes to which microtubules attach driving the separation and segregation of replicated sister chromosomes, have been viewed as essential for proper cell division and accurate transmission of chromosomes into daughter cells. However previous studies demonstrated that sister chromosomes lacking kinetochores (acentrics) often undergo separation, segregation and transmission. Here we demonstrate that sister acentrics are held together through DNA intertwining. We show that during anaphase, acentric sister separation is achieved through Topoisomerase activity, an enzyme that resolves these DNA linkages, as well as forces generated on the acentrics by the growing ends of highly dynamic microtubule polymers. We found that acentric sister chromatids display unique patterns of separation using mechanisms independent of the kinetochore. Additionally, we identified the specific microtubule-associated proteins required for the successful mitotic transmission of acentric chromosomes to daughter cells. These studies reveal unsuspected, distinct forces that likely act on all chromosomes during mitosis independent of kinetochore-microtubule attachments.

Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli.

Background: Co-expression of two distinct guide RNAs (gRNAs) has been used to facilitate the application of CRISPR/Cas9 system in fields such as large genomic deletion. The paired gRNAs are often placed adjacently in the same direction and expressed individually by two identical promoters, constituting direct repeats (DRs) which are susceptible to self-homologous recombination. As a result, the paired-gRNA plasmids cannot remain stable, which greatly prevents scalable application of the CRISPR/Cas9 system. Results: To address this limitation, different DRs-involved paired-gRNA plasmids were designed and the events of recombination were characterized. Deletion between DRs occurred with high frequencies during plasmid construction and subsequent plasmid propagation. This recombination event was RecA-independent, which agreed with the replication slippage model. To increase plasmid stability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs), which completely eliminated DRs-induced recombination. Using RPGPs, rapid deletion of chromosome fragments up to 100 kb with an efficiency of 83.33% was achieved in Escherichia coli. Conclusions: The RPGPs cloning strategy serves as a general solution to avoid plasmid RecA-independent recombination. It can be adapted to applications that rely on paired gRNAs or repeated genetic parts.