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Life Science Alliance Dec 2023The Dam1 complex is essential for mitotic progression across evolutionarily divergent fungi. Upon analyzing amino acid (aa) sequences of Dad2, a Dam1 complex subunit, we...
The Dam1 complex is essential for mitotic progression across evolutionarily divergent fungi. Upon analyzing amino acid (aa) sequences of Dad2, a Dam1 complex subunit, we identified a conserved 10-aa-long Dad2 signature sequence (DSS). An arginine residue (R126) in the DSS is essential for viability in that possesses point centromeres. The corresponding arginine residues are functionally important but not essential for viability in and ; both carry several kilobases long regional centromeres. The purified recombinant Dam1 complex containing either Dad2 or Dad2 failed to bind microtubules (MTs) or form any visible rings like the WT complex. Intriguingly, functional analysis revealed that the requirement of the conserved arginine residue for chromosome biorientation and mitotic progression reduced with increasing centromere length. We propose that plasticity of the invariant arginine of Dad2 in organisms with regional centromeres is achieved by conditional elevation of the kinetochore protein(s) to enable multiple kinetochore MTs to bind to each chromosome. The capacity of a chromosome to bind multiple kinetochore MTs may mask the deleterious effects of such lethal mutations.
Topics: Saccharomyces cerevisiae Proteins; Microtubule-Associated Proteins; Cell Cycle Proteins; Microtubules; Centromere; Saccharomyces cerevisiae; Arginine
PubMed: 37793775
DOI: 10.26508/lsa.202201720 -
Genes, Chromosomes & Cancer Nov 2023As the classification of kinase-driven spindle cell tumors continues to evolve, we describe the first series of pediatric mesenchymal tumors harboring FGFR1 gene fusions...
FGFR1 gene fusions in a subset of pediatric mesenchymal tumors: Expanding the genetic spectrum of tumors sharing histologic overlap with infantile fibrosarcoma and "NTRK-rearranged" spindle cell neoplasms.
As the classification of kinase-driven spindle cell tumors continues to evolve, we describe the first series of pediatric mesenchymal tumors harboring FGFR1 gene fusions that share histologic overlap with infantile fibrosarcoma and "NTRK-rearranged" spindle cell neoplasms. Herein, we present three cases of FGFR1-rearranged pediatric mesenchymal tumors, including one case with FGFR1::PARD6B gene fusion and two cases with FGFR1::EBF2 gene fusion. The tumors involved infants ranging from 3 to 9 months in age with a male-to-female ratio of 2:1. All tumors involved the deep soft tissue of the gluteal, pelvic, or perirectal region. Histologically, the tumors comprised a cellular spindle cell neoplasm with primitive stellate cells, focal myxoid stroma, focal epithelioid features, no necrosis, and occasional mitotic figures (2-6 per 10 high-power field). By immunohistochemistry, the neoplastic cells focally expressed CD34 but lacked expression of S100 protein, SMA, desmin, myogenin, MyoD1, pan-TRK, and ALK. These three cases, including a case with long-term clinical follow-up, demonstrate that FGFR1 fusions occur in a subset of newly described pediatric kinase-driven mesenchymal tumors with locally aggressive behavior. Importantly, knowledge of these genetic alterations in this spectrum of pediatric tumors is key for diagnostic and targeted therapeutic purposes.
Topics: Female; Humans; Infant; Male; Biomarkers, Tumor; Fibrosarcoma; Gene Fusion; Neoplasms, Connective and Soft Tissue; Receptor, Fibroblast Growth Factor, Type 1; Receptor, trkA; Soft Tissue Neoplasms
PubMed: 37265193
DOI: 10.1002/gcc.23179 -
Cell Genomics Nov 2023Chromosome-level design-build-test-learn cycles (chrDBTLs) allow systematic combinatorial reconfiguration of chromosomes with ease. Here, we established chrDBTL with a...
Chromosome-level design-build-test-learn cycles (chrDBTLs) allow systematic combinatorial reconfiguration of chromosomes with ease. Here, we established chrDBTL with a redesigned synthetic chromosome , . We designed and built to harbor strategically inserted features, modified elements, and synonymously recoded genes throughout the chromosome. Based on the recoded chromosome, we developed a method to enable chrDBTL: CRISPR-Cas9-mediated mitotic recombination with endoreduplication (CRIMiRE). CRIMiRE allowed the creation of customized wild-type/synthetic combinations, accelerating genotype-phenotype mapping and synthetic chromosome redesign. We also leveraged as a "build-to-learn" model organism for translation studies by ribosome profiling. We conducted a locus-to-locus comparison of ribosome occupancy between and the wild-type chromosome, providing insight into the effects of codon changes and redesigned features on translation dynamics . Overall, we established as a versatile reconfigurable system that advances chrDBTL for understanding biological mechanisms and engineering strains.
PubMed: 38020970
DOI: 10.1016/j.xgen.2023.100435 -
World Journal of Oncology Apr 2024Hepatocellular carcinoma (HCC) with high Ki67 protein expression, the most commonly used cell proliferation marker, is associated with an aggressive biologic phenotype;...
BACKGROUND
Hepatocellular carcinoma (HCC) with high Ki67 protein expression, the most commonly used cell proliferation marker, is associated with an aggressive biologic phenotype; however, conventional immunostaining is hampered by variability in institutional protocol, specific antibody probe, and by assessor subjectivity. To this end, we hypothesized that Ki67 gene () expression would identify highly proliferative HCC, and clarify its association with oncologic outcome, tumor progression, and immune cell population in the tumor microenvironment (TME). Furthermore, we sought to identify the cell-cycle gene expression profile that confers this aggressive phenotype.
METHODS
A total of 473 HCC patients with clinicopathological data associated with transcriptome were selected for this study: 358 patients from The Cancer Genome Atlas (TCGA) as the testing cohort, and 115 from GSE76427 as the validation cohort. Each cohort was divided into a highly proliferative group (MKi67-high) and the low MKi67 group (MKi67-low) by the median of Ki67 gene () expression levels.
RESULTS
MKi67-high HCC patients had worse disease-free survival (DFS), disease-specific survival (DSS), and overall survival (OS) independent of histological grade in the TCGA cohort. MKi67 expression correlated with histological grade and tumor size. MKi67 expression increased throughout the HCC carcinomatous sequence from normal liver, cirrhotic liver, early HCC, and advanced HCC. MKi67-high HCC was associated with higher intratumor heterogeneity, homologous recombination deficiency, and altered fraction as well as intratumoral infiltration of T helper type 1 (Th1) and Th2 cells, but lower interferon-gamma response and M2 macrophage infiltration. Cell proliferation-related gene sets in the Hallmark collection (E2F targets, G2M checkpoint, Myc target v1 and mitotic spindle), MTORC1 signaling, DNA repair, PI3K MTOR signaling, and unfolded protein response were all enriched in the MKi67-high HCC (false discovery rate (FDR) < 0.25).
CONCLUSIONS
High gene expression identified highly proliferative HCC with aggressive biology involving classical pathways in cell cycle regulation and DNA repair, as well as poor overall oncologic outcomes. This suggests potential for personalized treatment strategies, but validation and refinement of these observations require further research to elucidate the underlying mechanisms and validate therapeutic targeting of these pathways in MKi67-high HCC tumors.
PubMed: 38545476
DOI: 10.14740/wjon1751 -
Nature Communications Mar 2024DNA double-strand breaks (DSBs) are repaired by a hierarchically regulated network of pathways. Factors influencing the choice of particular repair pathways, however...
DNA double-strand breaks (DSBs) are repaired by a hierarchically regulated network of pathways. Factors influencing the choice of particular repair pathways, however remain poorly characterized. Here we develop an Integrated Classification Pipeline (ICP) to decompose and categorize CRISPR/Cas9 generated mutations on genomic target sites in complex multicellular insects. The ICP outputs graphic rank ordered classifications of mutant alleles to visualize discriminating DSB repair fingerprints generated from different target sites and alternative inheritance patterns of CRISPR components. We uncover highly reproducible lineage-specific mutation fingerprints in individual organisms and a developmental progression wherein Microhomology-Mediated End-Joining (MMEJ) or Insertion events predominate during early rapid mitotic cell cycles, switching to distinct subsets of Non-Homologous End-Joining (NHEJ) alleles, and then to Homology-Directed Repair (HDR)-based gene conversion. These repair signatures enable marker-free tracking of specific mutations in dynamic populations, including NHEJ and HDR events within the same samples, for in-depth analysis of diverse gene editing events.
Topics: DNA Breaks, Double-Stranded; Alleles; DNA Repair; DNA; DNA End-Joining Repair; Mutation; Recombinational DNA Repair; CRISPR-Cas Systems
PubMed: 38521791
DOI: 10.1038/s41467-024-46479-2 -
Methods in Molecular Biology (Clifton,... 2024Cytoskeletal motor proteins are essential molecular machines that hydrolyze ATP to generate force and motion along cytoskeletal filaments. Members of the dynein and...
Cytoskeletal motor proteins are essential molecular machines that hydrolyze ATP to generate force and motion along cytoskeletal filaments. Members of the dynein and kinesin superfamilies play critical roles in transporting biological payloads (such as proteins, organelles, and vesicles) along microtubule pathways, cause the beating of flagella and cilia, and act within the mitotic and meiotic spindles to segregate replicated chromosomes to progeny cells. Understanding the underlying mechanisms and behaviors of motor proteins is critical to provide better strategies for the treatment of motor protein-related diseases. Here, we provide detailed protocols for the recombinant expression of the Kinesin-1 motor KIF5C using a baculovirus/insect cell system and provide updated protocols for performing single-molecule studies using total internal reflection fluorescence microscopy and optical tweezers to study the motility and force generation of the purified motor.
Topics: Kinesins; Cytoskeletal Proteins; Microtubules; Spindle Apparatus; Dyneins
PubMed: 37824000
DOI: 10.1007/978-1-0716-3377-9_4 -
Journal of Microbiology (Seoul, Korea) Nov 2023Meiosis is a process through which diploid cells divide into haploid cells, thus promoting genetic diversity. This diversity arises from the formation of genetic...
Meiosis is a process through which diploid cells divide into haploid cells, thus promoting genetic diversity. This diversity arises from the formation of genetic crossovers (COs) that repair DNA double-strand breaks (DSBs), through homologous recombination (HR). Deficiencies in HR can lead to chromosomal abnormality resulting from chromosomal nondisjunction, and genetic disorders. Therefore, investigating the mechanisms underlying effective HR is crucial for reducing genome instability. Budding yeast serves as an ideal model for studying HR mechanisms due to its amenability to gene modifications and the ease of inducing synchronized meiosis to yield four spores. During meiosis, at the DNA level, programmed DSBs are repaired as COs or non-crossovers (NCOs) through structural alterations in the nascent D-loop, involving single-end invasions (SEIs) and double-Holliday junctions (dHJs). This repair occurs using homologous templates rather than sister templates. This protocol, using Southern blotting, allows for the analysis and monitoring of changes in DNA structures in the recombination process. One-dimensional (1D) gel electrophoresis is employed to detect DSBs, COs, and NCOs, while two-dimensional (2D) gel electrophoresis is utilized to identify joint molecules (JMs). Therefore, physical analysis is considered the most effective method for investigating the HR mechanism. Our protocol provides more comprehensive information than previous reports by introducing conditions for obtaining a greater number of cells from synchronized yeast and a method that can analyze not only meiotic/mitotic recombination but also mitotic replication.
Topics: Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; DNA Breaks, Double-Stranded; Meiosis; Homologous Recombination; DNA
PubMed: 38082069
DOI: 10.1007/s12275-023-00094-w -
Experimental Cell Research Sep 2023Exposure of eukaryotic cells to ionizing radiation or clastogenic chemicals leads to formation of DNA double-strand breaks (DSBs). These lesions are also generated...
Exposure of eukaryotic cells to ionizing radiation or clastogenic chemicals leads to formation of DNA double-strand breaks (DSBs). These lesions are also generated internally by chemicals and enzymes, in the absence of exogenous agents, though the sources and consequences of such endogenously generated DSBs remain poorly understood. In the current study, we have investigated the impact of reduced recombinational repair of endogenous DSBs on stress responses, cell morphology and other physical properties of S. cerevisiae (budding yeast) cells. Use of phase contrast and DAPI-based fluorescence microscopy combined with FACS analysis confirmed that recombination-deficient rad52 cell cultures exhibit chronically high levels of G phase cells. Cell cycle phase transit times during G, S and M were similar in WT and rad52 cells, but the length of G phase was increased by three-fold in the mutants. rad52 cells were larger than WT in all phases of the cycle and displayed other quantifiable changes in physical characteristics. The high G cell phenotype was abolished when DNA damage checkpoint genes, but not spindle assembly checkpoint genes, were co-inactivated with RAD52. Several other RAD52 group mutants (rad51, rad54, rad55, rad57 and rad59) also exhibited the high G cell phenotype. The results indicate that recombination deficiency leads to accumulation of unrepaired DSBs during normal mitotic growth that activate a major stress response and produce distinct changes in cellular physiology and morphology.
Topics: Saccharomyces cerevisiae; DNA-Binding Proteins; Saccharomyces cerevisiae Proteins; Rad51 Recombinase; DNA Repair; Cell Cycle; Homologous Recombination
PubMed: 37393982
DOI: 10.1016/j.yexcr.2023.113701 -
BMC Biology May 2024Sex-limited chromosomes Y and W share some characteristics, including the degeneration of protein-coding genes, enrichment of repetitive elements, and heterochromatin....
BACKGROUND
Sex-limited chromosomes Y and W share some characteristics, including the degeneration of protein-coding genes, enrichment of repetitive elements, and heterochromatin. However, although many studies have suggested that Y chromosomes retain genes related to male function, far less is known about W chromosomes and whether they retain genes related to female-specific function.
RESULTS
Here, we built a chromosome-level genome assembly of the Asian corn borer, Ostrinia furnacalis Guenée (Lepidoptera: Crambidae, Pyraloidea), an economically important pest in corn, from a female, including both the Z and W chromosome. Despite deep conservation of the Z chromosome across Lepidoptera, our chromosome-level W assembly reveals little conservation with available W chromosome sequence in related species or with the Z chromosome, consistent with a non-canonical origin of the W chromosome. The W chromosome has accumulated significant repetitive elements and experienced rapid gene gain from the remainder of the genome, with most genes exhibiting pseudogenization after duplication to the W. The genes that retain significant expression are largely enriched for functions in DNA recombination, the nucleosome, chromatin, and DNA binding, likely related to meiotic and mitotic processes within the female gonad.
CONCLUSIONS
Overall, our chromosome-level genome assembly supports the non-canonical origin of the W chromosome in O. furnacalis, which experienced rapid gene gain and loss, with the retention of genes related to female-specific function.
Topics: Animals; Moths; Female; Sex Chromosomes; Chromosomes, Insect; Male; Evolution, Molecular; Genome, Insect
PubMed: 38693535
DOI: 10.1186/s12915-024-01902-4 -
Genes, Chromosomes & Cancer Oct 2023Herein we report a case of an intraosseous myoepithelial carcinoma harboring a EWSR1::PBX3 fusion gene. The patient was a 64-year-old male found to have a 7 cm...
Herein we report a case of an intraosseous myoepithelial carcinoma harboring a EWSR1::PBX3 fusion gene. The patient was a 64-year-old male found to have a 7 cm destructive lesion in the distal ulna with an extraosseous soft tissue component. Microscopic examination of the resected tumor showed a spindle-cell lesion within a sclerotic stroma and intravascular tumor emboli. At higher power the tumor cells showed moderate nuclear atypia with a high mitotic count (20 per mm ). Immunohistochemistry revealed diffuse EMA positivity and focal pancytokeratin (AE1/AE3) and S100 expression, consistent with myoepithelial differentiation. NGS using the Oncomine Childhood Cancer Assay (Thermo Fisher Scientific, Inc.) revealed a EWSR1-PBX3 fusion and ABL amplification. The patient subsequently developed local recurrence as well as distant lymph node, lung and vertebral metastases; he is currently awaiting systemic treatment in the context of a clinical trial. In this report, we present a rare case of a skeletal myoepithelial tumor harboring a EWSR1::PBX3 fusion with demonstrated histological and clinical features of malignancy.
Topics: Humans; Male; Middle Aged; Biomarkers, Tumor; Bone Neoplasms; Carcinoma; Gene Fusion; Myoepithelioma; Neoplasms, Connective and Soft Tissue; RNA-Binding Protein EWS
PubMed: 37129228
DOI: 10.1002/gcc.23148