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1.
PLoS One ; 15(11): e0242506, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33226998

RESUMO

Microsatellites are widely applied in population and forensic genetics, wildlife studies and parentage testing in animal breeding, among others, and recently, high-throughput sequencing technologies have greatly facilitated the identification of microsatellite markers. In this study the genomic data of Cervus elaphus (CerEla1.0) was exploited, in order to identify microsatellite loci along the red deer genome and for designing the cognate primers. The bioinformatics pipeline identified 982,433 microsatellite motifs genome-wide, assorted along the chromosomes, from which 45,711 loci mapped to the X- and 1096 to the Y-chromosome. Primers were successfully designed for 170,873 loci, and validated with an independently developed autosomal tetranucleotide STR set. Ten X- and five Y-chromosome-linked microsatellites were selected and tested by two multiplex PCR setups on genomic DNA samples of 123 red deer stags. The average number of alleles per locus was 3.3, and the average gene diversity value of the markers was 0.270. The overall observed and expected heterozygosities were 0.755 and 0.832, respectively. Polymorphic Information Content (PIC) ranged between 0.469 and 0.909 per locus with a mean value of 0.813. Using the X- and Y-chromosome linked markers 19 different Y-chromosome and 72 X-chromosome lines were identified. Both the X- and the Y-haplotypes split to two distinct clades each. The Y-chromosome clades correlated strongly with the geographic origin of the haplotypes of the samples. Segregation and admixture of subpopulations were demonstrated by the use of the combination of nine autosomal and 16 sex chromosomal STRs concerning southwestern and northeastern Hungary. In conclusion, the approach demonstrated here is a very efficient method for developing microsatellite markers for species with available genomic sequence data, as well as for their use in individual identifications and in population genetics studies.


Assuntos
Cervos/genética , Repetições de Microssatélites/genética , Alelos , Animais , Cromossomos/genética , DNA/genética , Primers do DNA/genética , Feminino , Frequência do Gene/genética , Variação Genética/genética , Genoma/genética , Genótipo , Haplótipos/genética , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Masculino , Reação em Cadeia da Polimerase Multiplex/métodos , Cromossomo X/genética , Cromossomo Y/genética
2.
Cytogenet Genome Res ; 160(10): 610-624, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33207346

RESUMO

Agamid lizards (Squamata: Agamidae) are karyotypically heterogeneous. Among the 101 species currently described from Australia, all are from the subfamily Amphibolurinae. This group is, with some exceptions, karyotypically conserved, and all species involving heterogametic sex show female heterogamety. Here, we describe the chromosomes of 2 additional Australian agamid lizards, Tympanocryptis lineata and Rankinia diemensis. These species are phylogenetically and cytogenetically sisters to the well-characterised Pogona vitticeps, but their sex chromosomes and other chromosomal characteristics are unknown. In this study, we applied advanced molecular cytogenetic techniques, such as fluorescence in situ hybridisation (FISH) and cross-species gene mapping, to characterise chromosomes and to identify sex chromosomes in these species. Our data suggest that both species have a conserved karyotype with P. vitticeps but with subtle rearrangements in the chromosomal landscapes. We could identify that T. lineata possesses a female heterogametic system (ZZ/ZW) with a pair of sex microchromosomes, while R. diemensis may have heterogametic sex chromosomes, but this requires further investigations. Our study shows the pattern of chromosomal rearrangements between closely related species, explaining the speciation within Australian agamid lizards of similar karyotypes.


Assuntos
Cromossomos/genética , Rearranjo Gênico/genética , Cariótipo , Lagartos/genética , Animais , Bandeamento Cromossômico , Mapeamento Cromossômico , Cromossomos Artificiais Bacterianos/genética , Células Clonais , Metilação de DNA/genética , Geografia , Repetições de Microssatélites/genética , Especificidade da Espécie , Telômero/genética
3.
Sheng Wu Gong Cheng Xue Bao ; 36(10): 2040-2050, 2020 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-33169569

RESUMO

Linear chromatin is compacted into eukaryotic nucleus through a complex and multi-layered architecture. Consequently, chromatin conformation in a local or long-distance manner is strongly correlated with gene expression. Chromosome conformation capture (3C) technology, together with its variants like 4C/5C/Hi-C, has been well developed to study chromatin looping and whole genome structure. In this review, we introduce new technologies including chromosome capture combined with immunoprecipitation, nuclei acid-based hybridization, single cell and genome sequencing, as well as their application.


Assuntos
Cromatina , Cromossomos , Técnicas Genéticas , Núcleo Celular , Cromatina/genética , Cromossomos/química , Cromossomos/genética , Genoma/genética
4.
Nat Commun ; 11(1): 5795, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33199682

RESUMO

Chromosomes of all species studied so far display a variety of higher-order organisational features, such as self-interacting domains or loops. These structures, which are often associated to biological functions, form distinct, visible patterns on genome-wide contact maps generated by chromosome conformation capture approaches such as Hi-C. Here we present Chromosight, an algorithm inspired from computer vision that can detect patterns in contact maps. Chromosight has greater sensitivity than existing methods on synthetic simulated data, while being faster and applicable to any type of genomes, including bacteria, viruses, yeasts and mammals. Our method does not require any prior training dataset and works well with default parameters on data generated with various protocols.


Assuntos
Cromossomos/genética , Computadores , Reconhecimento Automatizado de Padrão , Algoritmos , Cromossomos Fúngicos/genética , Cromossomos Humanos/genética , Genoma Fúngico , Humanos , Saccharomyces cerevisiae/genética , Fluxo de Trabalho
5.
Cytogenet Genome Res ; 160(9): 539-553, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33227787

RESUMO

The family Aspredinidae comprises a clade of complex systematic relationships, both from molecular and morphological approaches. In this study, conventional and molecular cytogenetic studies coupled with nucleotide sequencing were performed in 6 Aspredininae species (Amaralia hypsiura, Bunocephalus cf. aloikae, Bunocephalus amaurus, Bunocephalus aff. coracoideus, Bunocephalus verrucosus, and Platystacus cotylephorus) from different locations of the Amazon hydrographic basin. Our results showed highly divergent diploid numbers (2n) among the species, ranging from 49 to 74, including the occurrence of an XX/X0 sex chromosome system. A neighbor-joining phylogram based on the cytochrome c oxidase I (COI) showed that Bunocephalus coracoideus is not a monophyletic clade, but closely related to B. verrucosus. The karyotypic data associated with COI suggest an ancestral karyotype for Aspredinidae with a reduced 2n, composed of bi-armed chromosomes and a trend toward chromosomal fissions resulting in higher diploid number karyotypes, mainly composed of acrocentric chromosomes. Evolutionary relationships were discussed under a phylogenetic context with related species from different Siluriformes families. The karyotype features and chromosomal diversity of Aspredinidae show an amazing differentiation, making this family a remarkable model for investigating the evolutionary dynamics in siluriforms as well as in fish as a whole.


Assuntos
Peixes-Gato/genética , Cromossomos/genética , Animais , Evolução Biológica , Brasil , Peixes-Gato/classificação , Cromossomos/ultraestrutura , Código de Barras de DNA Taxonômico , DNA Ribossômico/genética , Diploide , Evolução Molecular , Feminino , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Filogenia , RNA Ribossômico 18S/genética , RNA Ribossômico 5S/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Cromossomos Sexuais/genética , Cromossomos Sexuais/ultraestrutura , Especificidade da Espécie
6.
PLoS Genet ; 16(10): e1009155, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33119583

RESUMO

Chromosomes are likely to have assembled from unlinked genes in early evolution. Genetic linkage reduces the assortment load and intragenomic conflict in reproducing protocell models to the extent that chromosomes can go to fixation even if chromosomes suffer from a replicative disadvantage, relative to unlinked genes, proportional to their length. Here we numerically show that chromosomes spread within protocells even if recurrent deleterious mutations affecting replicating genes (as ribozymes) are considered. Dosage effect selects for optimal genomic composition within protocells that carries over to the genic composition of emerging chromosomes. Lacking an accurate segregation mechanism, protocells continue to benefit from the stochastic corrector principle (group selection of early replicators), but now at the chromosome level. A remarkable feature of this process is the appearance of multigene families (in optimal genic proportions) on chromosomes. An added benefit of chromosome formation is an increase in the selectively maintainable genome size (number of different genes), primarily due to the marked reduction of the assortment load. The establishment of chromosomes is under strong positive selection in protocells harboring unlinked genes. The error threshold of replication is raised to higher genome size by linkage due to the fact that deleterious mutations affecting protocells metabolism (hence fitness) show antagonistic (diminishing return) epistasis. This result strengthens the established benefit conferred by chromosomes on protocells allowing for the fixation of highly specific and efficient enzymes.


Assuntos
Cromossomos/genética , Evolução Molecular , Ligação Genética/genética , RNA Catalítico/genética , Replicação do DNA/genética , Epistasia Genética/genética , Genoma/genética , RNA/genética
7.
Sci Rep ; 10(1): 15993, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009476

RESUMO

The mating of 77 heterozygous pairs (Cav3.2[+|-] x Cav3.2[+|-]) revealed a significant deviation of genotype distribution from Mendelian inheritance in weaned pups. The mating of 14 pairs (Cav3.2[-|-] female x Cav3.2[+|-] male) and 8 pairs (Cav3.2[+|-] female x Cav3.2[-|-] male) confirmed the significant reduction of deficient homozygous Cav3.2[-|-] pups, leading to the conclusion that prenatal lethality may occur, when one or both alleles, encoding the Cav3.2T-type Ca2+ channel, are missing. Also, the mating of 63 heterozygous pairs (Cav2.3[+|-] x Cav2.3[+|-]) revealed a significant deviation of genotype distribution from Mendelian inheritance in weaned pups, but only for heterozygous male mice, leading to the conclusion that compensation may only occur for Cav2.3[-|-] male mice lacking both alleles of the R-type Ca2+ channel. During the mating of heterozygous parents, the number of female mice within the weaned population does not deviate from the expected Mendelian inheritance. During prenatal development, both, T- and R-type Ca2+ currents are higher expressed in some tissues than postnatally. It will be discussed that the function of voltage-gated Ca2+ channels during prenatal development must be investigated in more detail, not least to understand devastative diseases like developmental epileptic encephalopathies (DEE).


Assuntos
Canais de Cálcio Tipo R/fisiologia , Canais de Cálcio Tipo T/fisiologia , Proteínas de Transporte de Cátions/fisiologia , Cromossomos/genética , Instabilidade Genômica , Endogamia/métodos , Locos de Características Quantitativas , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
8.
Sci Rep ; 10(1): 17137, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-33051545

RESUMO

Complete hydatidiform moles (CHMs) comprise a proliferative trophoblastic disorder and are known to be androgenetic and diploid. Androgenetic CHMs are classified as having monospermic and dispermic origins. Rarely, some CHMs have other genetic constitutions, such as biparental diploid or tetraploid. Previous studies have shown the possibility that androgenetic heterozygous CHMs have an additional chromosome with high frequency. This study aimed to comprehensively analyse the molecular karyotyping of androgenetic dispermic CHMs and the parental contribution of their additional chromosomes. Single-nucleotide polymorphism arrays were performed with the genomic DNA of CHMs and patients. The B allele frequency and selected B allele frequency plotting of CHM were visualised. Among the 31 dispermic CHMs, eight showed trisomy and one showed double trisomy; of the 10 additional chromosomes, seven were of maternal original and three were of paternal origin. In addition, three disomic chromosomes comprised one maternal and one paternal chromosome, although these should theoretically have had two paternal chromosomes in the case of androgenetic CHMs. The subclassification of heterozygous CHMs, with or without maternal contribution, is a new approach and could be a candidate indicator of gestational trophoblastic neoplasia risk.


Assuntos
Androgênios/metabolismo , Mola Hidatiforme/genética , Trissomia/genética , Alelos , Animais , Cromossomos/genética , Diploide , Feminino , Genótipo , Heterozigoto , Humanos , Mola Hidatiforme/metabolismo , Masculino , Pais , Polimorfismo de Nucleotídeo Único/genética , Gravidez , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismo
9.
Proc Natl Acad Sci U S A ; 117(43): 26749-26755, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33051295

RESUMO

Spatial patterns are ubiquitous in both physical and biological systems. We have recently discovered that mitotic chromosomes sequentially acquire two interesting morphological patterns along their structural axes [L. Chu et al., Mol. Cell, 10.1016/j.molcel.2020.07.002 (2020)]. First, axes of closely conjoined sister chromosomes acquire regular undulations comprising nearly planar arrays of sequential half-helices of similar size and alternating handedness, accompanied by periodic kinks. This pattern, which persists through all later stages, provides a case of the geometric form known as a "perversion." Next, as sister chromosomes become distinct parallel units, their individual axes become linked by bridges, which are themselves miniature axes. These bridges are dramatically evenly spaced. Together, these effects comprise a unique instance of spatial patterning in a subcellular biological system. We present evidence that axis undulations and bridge arrays arise by a single continuous mechanically promoted progression, driven by stress within the chromosome axes. We further suggest that, after sister individualization, this same stress also promotes chromosome compaction by rendering the axes susceptible to the requisite molecular remodeling. Thus, by this scenario, the continuous presence of mechanical stress within the chromosome axes could potentially underlie the entire morphogenetic chromosomal program. Direct analogies with meiotic chromosomes suggest that the same effects could underlie interactions between homologous chromosomes as required for gametogenesis. Possible mechanical bases for generation of axis stress and resultant deformations are discussed. Together, these findings provide a perspective on the macroscopic changes of organized chromosomes.


Assuntos
Cromatina/química , Cromossomos/química , Mitose/genética , Morfogênese/genética , Linhagem Celular , Cromátides/química , Cromátides/genética , Cromátides/metabolismo , Cromatina/genética , Cromatina/metabolismo , Cromossomos/genética , Cromossomos/metabolismo , Humanos
10.
PLoS One ; 15(10): e0240935, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33119641

RESUMO

Sockeye salmon (Oncorhynchus nerka) is a commercially and culturally important species to the people that live along the northern Pacific Ocean coast. There are two main sockeye salmon ecotypes-the ocean-going (anadromous) ecotype and the fresh-water ecotype known as kokanee. The goal of this study was to better understand the population structure of sockeye salmon and identify possible genomic differences among populations and between the two ecotypes. In pursuit of this goal, we generated the first reference sockeye salmon genome assembly and an RNA-seq transcriptome data set to better annotate features of the assembly. Resequenced whole-genomes of 140 sockeye salmon and kokanee were analyzed to understand population structure and identify genomic differences between ecotypes. Three distinct geographic and genetic groups were identified from analyses of the resequencing data. Nucleotide variants in an immunoglobulin heavy chain variable gene cluster on chromosome 26 were found to differentiate the northwestern group from the southern and upper Columbia River groups. Several candidate genes were found to be associated with the kokanee ecotype. Many of these genes were related to ammonia tolerance or vision. Finally, the sex chromosomes of this species were better characterized, and an alternative sex-determination mechanism was identified in a subset of upper Columbia River kokanee.


Assuntos
Perfilação da Expressão Gênica/veterinária , Cadeias Pesadas de Imunoglobulinas/genética , Salmão/genética , Sequenciamento Completo do Genoma/veterinária , Animais , Cromossomos/genética , Ecótipo , Proteínas de Peixes/genética , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala/veterinária , Salmão/classificação , Análise de Sequência de RNA/veterinária
11.
Nat Genet ; 52(10): 1076-1087, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32868908

RESUMO

Animal chromosomes are partitioned into contact domains. Pathogenic domain disruptions can result from chromosomal rearrangements or perturbation of architectural factors. However, such broad-scale alterations are insufficient to define the minimal requirements for domain formation. Moreover, to what extent domains can be engineered is just beginning to be explored. In an attempt to create contact domains, we inserted a 2-kb DNA sequence underlying a tissue-invariant domain boundary-containing a CTCF-binding site (CBS) and a transcription start site (TSS)-into 16 ectopic loci across 11 chromosomes, and characterized its architectural impact. Depending on local constraints, this fragment variably formed new domains, partitioned existing ones, altered compartmentalization and initiated contacts reflecting chromatin loop extrusion. Deletions of the CBS or the TSS individually or in combination within inserts revealed its distinct contributions to genome folding. Altogether, short DNA insertions can suffice to shape the spatial genome in a manner influenced by chromatin context.


Assuntos
Fator de Ligação a CCCTC/genética , Cromatina/genética , Cromossomos/genética , Sítio de Iniciação de Transcrição , Animais , Sequência de Bases/genética , Sítios de Ligação/genética , Proteínas de Ligação a DNA/genética , Genoma/genética , Genoma Humano/genética , Humanos , Ligação Proteica/genética , Domínios Proteicos/genética
12.
PLoS One ; 15(8): e0238322, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32866178

RESUMO

Space-filling curves have been used for decades to study the folding principles of globular proteins, compact polymers, and chromatin. Formally, space-filling curves trace a single circuit through a set of points (x,y,z); informally, they correspond to a polymer melt. Although not quite a melt, the folding principles of Human chromatin are likened to the Hilbert curve: a type of space-filling curve. Hilbert-like curves in general make biologically compelling models of chromatin; in particular, they lack knots which facilitates chromatin folding, unfolding, and easy access to genes. Knot complexity has been intensely studied with the aid of Alexander polynomials; however, the approach does not generalize well to cases of more than one chromosome. Crossing complexity is an understudied alternative better suited for quantifying entanglement between chromosomes. Do Hilbert-like configurations limit crossing complexity between chromosomes? How does crossing complexity for Hilbert-like configurations compare to equilibrium configurations? To address these questions, we extend the Mansfield algorithm to enable sampling of Hilbert-like space filling curves on a simple cubic lattice. We use the extended algorithm to generate equilibrium, intermediate, and Hilbert-like configurational ensembles and compute crossing complexity between curves (chromosomes) in each configurational snapshot. Our main results are twofold: (a) Hilbert-like configurations limit entanglement between chromosomes and (b) Hilbert-like configurations do not limit entanglement in a model of S-phase DNA. Our second result is particularly surprising yet easily rationalized with a geometric argument. We explore ergodicity of the extended algorithm and discuss our results in the context of more sophisticated models of chromatin.


Assuntos
DNA/química , DNA/genética , Fase S/genética , Algoritmos , Cromatina/química , Cromatina/genética , Cromossomos/química , Cromossomos/genética , Humanos , Polímeros/química
13.
Nat Commun ; 11(1): 4432, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887874

RESUMO

Spontaneous coronary artery dissection (SCAD) is a non-atherosclerotic cause of myocardial infarction (MI), typically in young women. We undertook a genome-wide association study of SCAD (Ncases = 270/Ncontrols = 5,263) and identified and replicated an association of rs12740679 at chromosome 1q21.2 (Pdiscovery+replication = 2.19 × 10-12, OR = 1.8) influencing ADAMTSL4 expression. Meta-analysis of discovery and replication samples identified associations with P < 5 × 10-8 at chromosome 6p24.1 in PHACTR1, chromosome 12q13.3 in LRP1, and in females-only, at chromosome 21q22.11 near LINC00310. A polygenic risk score for SCAD was associated with (1) higher risk of SCAD in individuals with fibromuscular dysplasia (P = 0.021, OR = 1.82 [95% CI: 1.09-3.02]) and (2) lower risk of atherosclerotic coronary artery disease and MI in the UK Biobank (P = 1.28 × 10-17, HR = 0.91 [95% CI :0.89-0.93], for MI) and Million Veteran Program (P = 9.33 × 10-36, OR = 0.95 [95% CI: 0.94-0.96], for CAD; P = 3.35 × 10-6, OR = 0.96 [95% CI: 0.95-0.98] for MI). Here we report that SCAD-related MI and atherosclerotic MI exist at opposite ends of a genetic risk spectrum, inciting MI with disparate underlying vascular biology.


Assuntos
Anomalias dos Vasos Coronários/genética , Genes Neoplásicos , Infarto do Miocárdio/genética , Doenças Vasculares/congênito , Proteínas ADAMTS/genética , Doenças das Artérias Carótidas/complicações , Doenças das Artérias Carótidas/genética , Cromossomos/genética , Estudos de Coortes , Doença da Artéria Coronariana/genética , Feminino , Displasia Fibromuscular/complicações , Displasia Fibromuscular/genética , Estudo de Associação Genômica Ampla , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Masculino , Metanálise como Assunto , Proteínas dos Microfilamentos/genética , Fatores de Risco , Doenças Vasculares/genética
14.
PLoS One ; 15(9): e0239450, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32970714

RESUMO

BACKGROUND: Bone abnormality and leg disease in commercial broiler flocks are increasingly prominent, causing serious economic losses to the broiler breeding industry. Valgus-varus deformity (VVD) is a common deformity of the long bone in broilers that manifests as an outward or inward deviation of the tibiotarsus or tarsometatarsus. There is a paucity of studies on the molecular mechanisms of VVD. RESULTS: In this study, 6 cDNA libraries were constructed from spleen samples from VVD birds and normal birds. A total of 1951 annotated lncRNAs, 7943 novel lncRNAs and 30252 mRNAs were identified by RNA-sequencing. In addition, 420 differentially expressed (DE) mRNAs and 124 differentially expressed lncRNAs (adjusted P-value < 0.05) were obtained. A total of 16 dysregulated genes were confirmed by qPCR to be consistent with the results of the RNA-Seq. The functional lncRNA-mRNA co-expression network was constructed using differentially expressed mRNAs and target genes of the differentially expressed lncRNAs. 11 DE genes were obtained from the analysis. In order to gain insight into the interactions of genes, lncRNAs and pathways associated with VVD, we focused on the following pathways, which are involved in immunity and bone development: the Jak-stat signaling pathway, Toll-like receptor signaling pathway, Wnt-signaling pathway, mTOR signaling pathway, VEGF signaling pathway, Notch signaling pathway, TGF-beta signaling pathway and Fanconi anemia pathway. All together, 30 candidate DE genes were obtained from these pathways. We then analyzed the interaction between the DE genes and their corresponding lncRNAs. From these interaction network analyses we found that GARS, NFIC, PIK3R1, BMP6, NOTCH1, ACTB and CREBBP were the key core nodes of these networks. CONCLUSION: This study showed that differentially expressed genes and signaling pathways were related to immunity or bone development. These results increase the understanding of the molecular mechanisms of VVD and provide some reference for the etiology and pathogenesis of VVD.


Assuntos
Doenças Ósseas/genética , Doenças das Aves Domésticas/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Animais , Desenvolvimento Ósseo/genética , Doenças Ósseas/patologia , Galinhas/genética , Cromossomos/genética , Análise por Conglomerados , Regulação para Baixo , Biblioteca Gênica , Redes Reguladoras de Genes , Doenças das Aves Domésticas/patologia , RNA Longo não Codificante/química , RNA Mensageiro/química , Análise de Sequência de RNA , Baço/metabolismo , Transcriptoma , Regulação para Cima
15.
Nucleic Acids Res ; 48(18): 10353-10367, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32926139

RESUMO

The vast majority of the genome is transcribed by RNA polymerases. G+C-rich regions of the chromosomes and negative superhelicity can promote the invasion of the DNA by RNA to form R-loops, which have been shown to block DNA replication and promote genome instability. However, it is unclear whether the R-loops themselves are sufficient to cause this instability or if additional factors are required. We have investigated replisome collisions with transcription complexes and R-loops using a reconstituted bacterial DNA replication system. RNA polymerase transcription complexes co-directionally oriented with the replication fork were transient blockages, whereas those oriented head-on were severe, stable blockages. On the other hand, replisomes easily bypassed R-loops on either template strand. Replication encounters with R-loops on the leading-strand template (co-directional) resulted in gaps in the nascent leading strand, whereas lagging-strand template R-loops (head-on) had little impact on replication fork progression. We conclude that whereas R-loops alone can act as transient replication blocks, most genome-destabilizing replication fork stalling likely occurs because of proteins bound to the R-loops.


Assuntos
Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Estruturas R-Loop/genética , Transcrição Genética , Composição de Bases/genética , Estruturas Cromossômicas/genética , Cromossomos/genética , DNA Helicases/genética , Reparo do DNA/genética , Escherichia coli/genética , Instabilidade Genômica/genética
16.
Mol Cell ; 80(2): 246-262.e4, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32949493

RESUMO

CRISPR-Cas9-based gene drive systems possess the inherent capacity to spread progressively throughout target populations. Here we describe two self-copying (or active) guide RNA-only genetic elements, called e-CHACRs and ERACRs. These elements use Cas9 produced in trans by a gene drive either to inactivate the cas9 transgene (e-CHACRs) or to delete and replace the gene drive (ERACRs). e-CHACRs can be inserted at various genomic locations and carry two or more gRNAs, the first copying the e-CHACR and the second mutating and inactivating the cas9 transgene. Alternatively, ERACRs are inserted at the same genomic location as a gene drive, carrying two gRNAs that cut on either side of the gene drive to excise it. e-CHACRs efficiently inactivate Cas9 and can drive to completion in cage experiments. Similarly, ERACRs, particularly those carrying a recoded cDNA-restoring endogenous gene activity, can drive reliably to fully replace a gene drive. We compare the strengths of these two systems.


Assuntos
Deleção de Genes , Tecnologia de Impulso Genético , Animais , Proteína 9 Associada à CRISPR/metabolismo , Cromossomos/genética , Drosophila melanogaster/genética , Feminino , Proteínas de Fluorescência Verde/metabolismo , Padrões de Herança/genética , Mutagênese/genética , RNA Guia/genética , Transgenes
17.
BMC Evol Biol ; 20(1): 113, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32883209

RESUMO

BACKGROUND: The study of speciation has expanded with the increasing availability and affordability of high-resolution genomic data. How the genome evolves throughout the process of divergence and which regions of the genome are responsible for causing and maintaining that divergence have been central questions in recent work. Here, we use three pairs of species from the recently diverged bee hummingbird clade to investigate differences in the genome at different stages of speciation, using divergence times as a proxy for the speciation continuum. RESULTS: Population measures of relative differentiation between hybridizing species reveal that different chromosome types diverge at different stages of speciation. Using FST as our relative measure of differentiation we found that the sex chromosome shows signs of divergence early in speciation. Next, small autosomes (microchromosomes) accumulate highly diverged genomic regions, while the large autosomes (macrochromosomes) accumulate genomic regions of divergence at a later stage of speciation. CONCLUSIONS: Our finding that genomic windows of elevated FST accumulate on small autosomes earlier in speciation than on larger autosomes is counter to the prediction that FST increases with size of chromosome (i.e. with decreased recombination rate), and is not represented when weighted average FST per chromosome is compared with chromosome size. The results of this study suggest that multiple chromosome characteristics such as recombination rate and gene density combine to influence the genomic locations of signatures of divergence.


Assuntos
Evolução Biológica , Aves/classificação , Especiação Genética , Animais , Cromossomos/genética , Genoma , Genômica , Hibridização Genética , Cromossomos Sexuais/genética
18.
PLoS Genet ; 16(9): e1009001, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886661

RESUMO

During meiosis, diploid organisms reduce their chromosome number by half to generate haploid gametes. This process depends on the repair of double strand DNA breaks as crossover recombination events between homologous chromosomes, which hold homologs together to ensure their proper segregation to opposite spindle poles during the first meiotic division. Although most organisms are limited in the number of crossovers between homologs by a phenomenon called crossover interference, the consequences of excess interfering crossovers on meiotic chromosome segregation are not well known. Here we show that extra interfering crossovers lead to a range of meiotic defects and we uncover mechanisms that counteract these errors. Using chromosomes that exhibit a high frequency of supernumerary crossovers in Caenorhabditis elegans, we find that essential chromosomal structures are mispatterned in the presence of multiple crossovers, subjecting chromosomes to improper spindle forces and leading to defects in metaphase alignment. Additionally, the chromosomes with extra interfering crossovers often exhibited segregation defects in anaphase I, with a high incidence of chromatin bridges that sometimes created a tether between the chromosome and the first polar body. However, these anaphase I bridges were often able to resolve in a LEM-3 nuclease dependent manner, and chromosome tethers that persisted were frequently resolved during Meiosis II by a second mechanism that preferentially segregates the tethered sister chromatid into the polar body. Altogether these findings demonstrate that excess interfering crossovers can severely impact chromosome patterning and segregation, highlighting the importance of limiting the number of recombination events between homologous chromosomes for the proper execution of meiosis.


Assuntos
Segregação de Cromossomos/genética , Troca Genética/genética , Meiose/genética , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Cromátides/genética , Cromatina/genética , Posicionamento Cromossômico/genética , Cromossomos/genética , Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/genética , Recombinação Genética
19.
Mol Cell ; 79(6): 902-916.e6, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32768407

RESUMO

A long-standing conundrum is how mitotic chromosomes can compact, as required for clean separation to daughter cells, while maintaining close parallel alignment of sister chromatids. Pursuit of this question, by high resolution 3D fluorescence imaging of living and fixed mammalian cells, has led to three discoveries. First, we show that the structural axes of separated sister chromatids are linked by evenly spaced "mini-axis" bridges. Second, when chromosomes first emerge as discrete units, at prophase, they are organized as co-oriented sister linear loop arrays emanating from a conjoined axis. We show that this same basic organization persists throughout mitosis, without helical coiling. Third, from prophase onward, chromosomes are deformed into sequential arrays of half-helical segments of alternating handedness (perversions), accompanied by correlated kinks. These arrays fluctuate dynamically over <15 s timescales. Together these discoveries redefine the foundation for thinking about the evolution of mitotic chromosomes as they prepare for anaphase segregation.


Assuntos
Proteínas de Ciclo Celular/genética , Cromossomos/genética , Proteínas de Ligação a DNA/genética , Mitose/genética , Adenosina Trifosfatases/genética , Anáfase/genética , Animais , Proteínas de Ciclo Celular/isolamento & purificação , Cromátides/genética , Proteínas Cromossômicas não Histona , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/isolamento & purificação , Imageamento Tridimensional , Mamíferos , Metáfase/genética , Prófase/genética
20.
Mol Cell ; 79(6): 881-901, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32768408

RESUMO

Nucleosomes package genomic DNA into chromatin. By regulating DNA access for transcription, replication, DNA repair, and epigenetic modification, chromatin forms the nexus of most nuclear processes. In addition, dynamic organization of chromatin underlies both regulation of gene expression and evolution of chromosomes into individualized sister objects, which can segregate cleanly to different daughter cells at anaphase. This collaborative review shines a spotlight on technologies that will be crucial to interrogate key questions in chromatin and chromosome biology including state-of-the-art microscopy techniques, tools to physically manipulate chromatin, single-cell methods to measure chromatin accessibility, computational imaging with neural networks and analytical tools to interpret chromatin structure and dynamics. In addition, this review provides perspectives on how these tools can be applied to specific research fields such as genome stability and developmental biology and to test concepts such as phase separation of chromatin.


Assuntos
Cromatina/genética , Cromossomos/genética , DNA/genética , Nucleossomos/genética , Reparo do DNA/genética , Replicação do DNA/genética , Epigênese Genética/genética , Humanos
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