Chromosome Research最新文献

The segmentation of overlapping chromosomes in metaphase images is a longstanding challenge in cytogenetics, where limited spectral contrast in conventional RGB imaging. In this study, we explore hyperspectral imaging (HSI) as a promising alternative modality to enhance segmentation accuracy by exploiting information from a narrow band of the spectrum. To evaluate the benefit of spectral data, we acquired and annotated a dedicated dataset of overlapping chromosomes, using a hyperspectral microscopy system. Multiple segmentation baseline models were trained and benchmarked on both RGB and HSI data. Experimental results show that HSI-based models consistently outperform RGB-based counterparts, achieving up to 92% Dice score. These findings underline the potential of HSI for improving cytogenetic image analysis and provide a new benchmark dataset for future research in spectral segmentation.

The ongoing development of human artificial chromosomes (HACs) will permit investigation into essential centromere processes and the means to deliver large genetic cargoes to target cells. Starting with large (~750 kb) yeast artificial chromosome (YAC)-based constructs limits the rampant multimerization that has complicated many prior types of HACs. Large YAC construction is accomplished using transformation-associated recombination (TAR) strategies that can become unwieldly when several functional modules are to be incorporated and tested. To address this issue, we developed an approach where modules are built using high-fidelity in vitro assembly strategies in a bacterial artificial chromosome (BAC) format. Then, the assembled modules are transferred in a simplified TAR step into a recipient YAC harboring the prokaryotic "stuffer" DNA that comprises a large portion of the final HAC construct. This approach is highly efficient with two-thirds of all screened yeast clones harboring the correct TAR product. Further, whole-genome Oxford Nanopore Technologies (ONT) sequencing/alignments, de novo assembly of the final YAC using a single ONT sequencing run, and close inspection of highly repetitive regions are all streamlined to rapidly validate clones that match the design. The fully sequenced, verified strain harboring a multi-module construct was then fused to human cells, where it efficiently formed functional HACs upon initial seeding with CENP-A-containing nucleosomes. We envision that the rapid assembly steps will be useful to quickly incorporate different functional modules, including diverse genetic cargoes, to engineer HACs with specific design features.

Species from the tribe Triticeae can be used for wheat improvement by introgression of valuable genes through wide hybridization. More than 85% of Triticeae genomes consist of repetitive sequences with species-specific content and composition, which can be used to detect introgression chromatin. We developed repeat-based molecular markers specific for the D genome of allopolyploid wheat Triticum aestivum (2n = 6x = 42, AABBDD) and applied them to detect D-genome chromatin in tetraploid wheat T. turgidum (2n = 4x = 28, AABB). To identify wheat subgenome-specific repeats, comparative analysis of low-coverage sequence data was performed using the RepeatExplorer pipeline. Three types of repeats, suitable for the development of D-genome-specific markers were identified: the centromeric retrotransposons Quinta, Afa-family repeats associated with the CACTA DNA transposons and the Ty3/gypsy LTR retrotransposon from the Tekay lineage. Consensus sequences of these repeats were used to select D-genome-specific SNPs and design PCR allelic competitive extension (PACE) assays for detecting D-genome chromatin. The assays were applied to screen introgression populations derived from T. aestivum x T. turgidum crosses. The PACE results were confirmed by fluorescent in situ hybridization, which showed that the markers detected introgressions ranging from translocation segments to intact chromosomes. Given the increasing availability of whole-genome sequence data, the presented approach can be used to develop molecular markers for detection of any introgression regardless of its chromosomal homoeologous group.

In the 40 years since the discovery of the CENP proteins, many studies have examined the role of these proteins and their interactions with other chromosomal proteins of the centromere and beyond. Together, these studies have yielded vast amounts of sequencing and proteomics data. Typically, each study has focused on a single question and the majority of each dataset remains largely unexplored. Often the interesting details of publicly deposited data are left behind, buried in archives online, while more and more new data are generated. Reanalysing these databases can represent a new paradigm for investigating diverse biological pathways in unprecedented detail. Here, we explore two publicly available pan-cancer proteomic datasets to compare proteins whose abundance correlates with CENP proteins, with a particular focus on CENP-C. Our analysis confirms an expected link between CENP-C and cohesin levels but reveals a surprising and unexpected correlation between CENP-C and proteins of the inner nuclear membrane and the NuMA protein. This guilt-by-association analysis has the potential to identify proteins that act in common pathways but never associate or colocalize and may not even be expressed at the same time in cells. As an example, we show here that it can reveal unexpected links that expand our conception of centromeric chromatin beyond chromosome segregation.

B chromosomes are nonessential chromosomes and are considered genetically inert. However, the maize B chromosome possesses several genetic properties during male gamete development in anthers. To determine whether the transcriptome of maize anthers is influenced by the B chromosome, we established the correlation between anther length and developmental stage in the maize B73 inbred line and applied complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) analysis to microsporogenetic (MS) anthers, microgametogenetic (MG) anthers and mature pollen (MP) of B73 plants carrying zero (0B) and two B chromosomes (2B). The results revealed that 6.4%, 7.4% and 25.1% of 0B-extra or 2B-extra cDNA-AFLP fragments from MS anthers, MG anthers and MP, respectively, are present between B73 + 0B and B73 + 2B. Twelve out of the 13 2B-extra cDNA-AFLP fragments from MS anthers and MP are A chromosome-located sequences and are transcribed in the presence of the B chromosome. Some of these sequences are functional genes. The remaining 2B-extra cDNA-AFLP fragment isolated from MS anthers is a novel B chromosome-specific repeat, pMS4. On the basis of three B-10L translocations and fluorescence in situ hybridization, pMS4 was mapped to the short arm and the junction between the centromeric knob and the proximal euchromatin of the B chromosome. Reverse transcriptase PCR revealed that pMS4 is transcribed in a B chromosome dosage-dependent and tissue-specific manner. Taken together, our data suggest that the maize B chromosome can transcribe and influence the transcription of A chromosome-located genes in anthers at different developmental stages.

Crepis sensu lato (s.l.) comprises species with remarkable variation in genome size, chromosome number, and karyotype structure, making this genus a valuable model for studying genome and chromosome evolution. Studies in various plant systems have suggested that diversification and speciation may be accompanied by dynamic changes in the repetitive DNA fraction, including satellite DNAs (satDNAs). A complex approach consisting of molecular (cloning, dot-blot and Southern hybridisation), cytogenetic (fluorescent in situ hybridisation) and phylogenetic methods allowed assessing the chromosomal and genomic organisation of three satDNAs in 32 species from Crepis s.l.. In the present study, we posed a question of whether these satDNAs are specific to C. capillaris, supporting the "birth-and-death" hypothesis, or whether they are also present in related species, consistent with the "library hypothesis". Each satDNA family exhibited different evolutionary trajectories. While pCcE9 amplification was specific to C. capillaris, supporting the "birth-and-death" hypothesis, two other analysed satDNAs were present in several related species, consistent with the "library hypothesis". Notably, pCcD29 showed different genomic and chromosomal organisation among C. capillaris and the species from the C. vesicaria group, suggesting that the satDNA evolution model proposed by Ruiz-Ruano et al. for animal systems may also apply to Crepis species. The organisation of the pCcD29 family ranges from numerous small, poorly homogenised loci (C. polymorpha) through organisation in both shorter and longer arrays (C. taraxacifolia) to a few major, highly homogenised loci (C. capillaris), highlighting contrasting evolutionary pathways of satDNAs within closely related species.

The phenotypic similarities and genetic heterogeneity occurring in diverse forms of Ehlers Danlos Syndrome (EDS) subtypes and many heritable connective tissue disorders can pose a diagnostic challenge. In the wake of the growing applications of next-generation sequencing technologies including exome and genome sequencing, opportunities for achieving definitive genetic diagnosis are increasingly arising. We present a 46-year-old man with joint laxity, recurrent joint subluxations, pelvic floor dysfunction, and postural orthostatic tachycardia syndrome (POTS), who was referred for EDS assessment. His medical history included morbid obesity requiring gastric bypass surgery, hearing loss, asthma, retinopathy, myopia, atrial septal defect, narcolepsy with cataplexy, polyneuropathy, folliculitis, lichen simplex chronicus, atopic dermatitis, and hypogonadism. His family history was significant for multiple first- and second-degree relatives who died from cardiac diseases including cases of childhood deaths. Physical examination showed joint laxity with Beighton score of 3/9, bilateral pes planus, hearing loss and macrocephaly. Exome sequencing revealed heterozygous variants LMNA c.1262 T > C p.L421P [classified as likely pathogenic], FLG c.2282_2285del p. S761Cfs*36 [classified as pathogenic], and FLG c.1501 C > T p. R501* [classified as pathogenic]. Mitochondria sequencing revealed a variant of uncertain significance (VUS), MT-ND2 m.5047 T > C p.V193A that is present at 9% heteroplasmy in blood. These findings show co-occurrence of pathogenic sequence variants in neighboring genes located in chromosome 1q2 region [LMNA and FLG] in a patient with features of hereditary connective tissue disorders. Our study highlights the capability of exome sequencing in achieving some actionable diagnosis in cases of co-morbid genetic disorders with overlapping and non-specific symptoms.

Centromeres have been the focus of extensive research for almost a century, so it may come as a surprise that a consistent definition and nomenclature for these structures remains elusive. In recent times, centromeric chromatin is most frequently defined by the presence of nucleosomes containing the H3 variant CENP-A and is typically synonymous with the site of the inner-kinetochore. However, crucial mammalian centromere proteins including CENP-B and INCENP have well defined distributions that show very little overlap with CENP-A. Additional protein localisations spanning the primary constriction or forming a band below CENP-A chromatin have been reported. Together, these observations suggest a complex and multi-layered chromatin organisation that is not well served by the canonical dichotomy of 'centromeric' and 'pericentromeric' chromatin. Strikingly, this is not a new observation but was made soon after the discovery of CENP proteins, including in a 1991 publication titled 'When is the centromere not a kinetochore?'. Here we revisit this question, which has become more pertinent following technical innovations in long-read sequencing and super-resolution microscopy. We present a model of centromere organisation for monocentromeres that incorporates additional complexity. We then use this model to reconceptualise diverse centromere forms in other eukaryotes including regional centromeres, holocentromeres and centromeres that lack key proteins including CENP-A. In this way, we hope to move towards a unified understanding of centromeric chromatin.

Centromeres are fundamental chromosomal structures that ensure accurate chromosome segregation during cell division. Despite their conserved and essential role in maintaining genomic stability, centromeres are subject to rapid evolutionary change. At the heart of centromere identity is the histone H3 variant CENP-A, an epigenetic mark that defines and propagates active centromeres and is essential for their function. Recent evidence supports a rapid evolution of centromere DNA sequences but also suggests a certain degree of flexibility in CENP-A deposition and propagation. The phenomenon of centromere drift, recently observed in humans, highlights how the dynamic repositioning of CENP-A and associated epigenetic environment over time maintains a regulated equilibrium, ensuring centromere function despite positional variation. Understanding these processes is crucial for unraveling centromere dynamics and their broader implications for genome stability and evolution.

Four decades ago, the discovery of centromere protein-A (CENP-A) marked a pivotal breakthrough in chromosome biology, revealing the epigenetic foundation of centromere identity. CENP-A, a histone H3 variant, directs the formation of the microtubule-binding kinetochore complex, designating the chromosomal site for its assembly and underpins the accurate partitioning of genetic material during cell division. Errors in cell division can give rise to DNA instability and aneuploidy, implicated in human diseases such as cancer. Therefore, discovering the underlying pathways and mechanisms responsible for the formation, regulation and maintenance of the centromere is important to our understanding of genome stability, epigenetic inheritance, and in providing the knowledge to help generate possible treatments and therapeutics. Here, we review various molecular pathways and mechanisms implicated in maintaining centromere identity and highlight some of the key outstanding questions with a focus on the human centromere.