The selfish yeast plasmid utilizes the condensin complex and condensed chromatin for faithful partitioning

Equipartitioning by chromosome association and copy number correction by DNA amplification are at the heart of the evolutionary success of the selfish yeast 2-micron plasmid. The present analysis reveals frequent plasmid presence near telomeres (TELs) and centromeres (CENs) in mitotic cells, with a preference towards the former. Inactivation of Cdc14 causes plasmid missegregation, which is correlated to the non-disjunction of TELs (and of rDNA) under this condition. Induced missegregation of chromosome XII, one of the largest yeast chromosomes which harbors the rDNA array and is highly dependent on the condensin complex for proper disjunction, increases 2-micron plasmid missegregation. This is not the case when chromosome III, one of the smallest chromosomes, is forced to missegregate. Plasmid stability decreases when the condensin subunit Brn1 is inactivated. Brn1 is recruited to the plasmid partitioning locus (STB) with the assistance of the plasmid-coded partitioning proteins Rep1 and Rep2. Furthermore, in a dihybrid assay, Brn1 interacts with Rep1-Rep2. Taken together, these findings support a role for condensin and/or condensed chromatin in 2-micron plasmid propagation. They suggest that condensed chromosome loci are among favored sites utilized by the plasmid for its chromosome-associated segregation. By homing to condensed/quiescent chromosome locales, and not over-perturbing genome homeostasis, the plasmid may minimize fitness conflicts with its host. Analogous persistence strategies may be utilized by other extrachromosomal selfish genomes, for example, episomes of mammalian viruses that hitchhike on host chromosomes for their stable maintenance.

Identification of potential and novel target genes in pituitary prolactinoma by bioinformatics analysis

Pituitary prolactinoma is one of the most complicated and fatally pathogenic pituitary adenomas. Therefore, there is an urgent need to improve our understanding of the underlying molecular mechanism that drives the initiation, progression, and metastasis of pituitary prolactinoma. The aim of the present study was to identify the key genes and signaling pathways associated with pituitary prolactinoma using bioinformatics analysis. Transcriptome microarray dataset GSE119063 was acquired from Gene Expression Omnibus datasets, which included 5 pituitary prolactinoma samples and 4 normal pituitaries samples. We screened differentially expressed genes (DEGs) with limma and investigated their biological function by pathway and Gene Ontology (GO) enrichment analysis. A protein-protein interaction (PPI) network of the up and down DEGs were constructed and analyzed by HIPPIE and Cytoscape software. Module analyses were performed. In addition, a target gene - miRNA network and target gene - TF network of the up and down DEGs were constructed by NetworkAnalyst and Cytoscape software. The set of DEGs exhibited an intersection consisting of 989 genes (461 up-regulated and 528 down-regulated), which may be associated with pituitary prolactinoma. Pathway enrichment analysis showed that the 989 DEGs were significantly enriched in the retinoate biosynthesis II, signaling pathways regulating pluripotency of stem cells, ALK2 signaling events, vitamin D3 biosynthesis, cell cycle and aurora B signaling. Gene Ontology (GO) enrichment analysis also showed that sensory organ morphogenesis, extracellular matrix, hormone activity, nuclear division, condensed chromosome and microtubule binding. In the PPI network and modules, SOX2, PRSS45, CLTC, PLK1, B4GALT6, RUNX1 and GTSE1 were considered as hub genes. In the target gene miRNA network and target gene - TF network, LINC00598, SOX4, IRX1 and UNC13A were considered as hub genes. Using integrated bioinformatics analysis, we identified candidate genes in pituitary prolactinoma, which may improve our understanding of the mechanisms of the pathogenesis and integration; genes may be therapeutic targets and prognostic markers for pituitary prolactinoma.

The Effect of Magnesium Ions on Chromosome Structure: Insights from G-Banding

Magnesium ion (Mg2+) plays a fundamental role in the chromosome condensation which is important for genetic material segregation. Studies about the effects of Mg2+ on the overall chromosome structure have been reported. Nevertheless, its effects on the distribution of heterochromatin and euchromatin region have yet to be investigated. This study was aimed to evaluate the effects of Mg2+ on the banding pattern of the chromosome structure. Chromosome analysis was performed using the GTL-banding technique on synchronized HeLa cells. The effect of Mg2+ was evaluated by subjecting the chromosomes to three different solutions, namely XBE5 (5 mM Mg2+) as a control, XBE (0 mM Mg2+), and 1 mM EDTA as cations chelator. The results showed a condensed chromosome structure with a clear banding pattern when it was treated with a buffer containing 5 mM Mg2+. In contrast, chromosomes treated with a buffer containing no Mg2+ and those treated with an ions chelator showed an expanded and fibrous structure with the lower intensity of the banding pattern. Elongation of the chromosome caused by decondensation resulted in the band splitting. The results of this study further emphasized the role of Mg2+ on chromosome structure and gave insights into Mg2+ effects on the banding distribution.

Identification of key genes and their microRNAs in glioma: Evidence from bioinformatic analysis

Background Glioma is one of the most common primary intracranial tumors. Although a lot of studies have been conducted to elucidate the pathogeny of glioma, the molecular mechanisms are still unclear because of its complex biological functions. Methods To identify the candidate genes in the carcinogenesis and progression of glioma, microarray datasets GSE4290, GSE122498 and GSE2223 were downloaded from Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were identified, and performed function enrichment of DEGs by Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The protein-protein interaction network (PPI) was constructed using STRING and Cytoscape to find hub genes. Survival analysis and GEPIA database was conducted to screen and validate critical genes. Analysis of miRNA and genetic alteration was used to explore and predict the molecule mechanism. Results A total of 150 DEGs were identified, consisting of 54 downregulated genes and 96 upregulated genes. The enriched functions and pathways of the DEGs include regulation of transportation, synaptic transmission and SH3 domain binding. Fifteen hub genes were identified and biological process analysis revealed that these genes were mainly enriched in SH3 domain binding, neuron projection terminus, mitotic nuclear division, condensed chromosome and affected the brain development. Survival analysis showed that VAMP2, PPP3CA, DLGAP5, KIF14, REPS2, CENPU, KNTC1 and SMC4, may be involved in the carcinogenesis, invasion or recurrence of glioma. These 8 hub genes, which were related miRNAs and genetic changes were commonly involved in the development of glioma, were closely associated with tumor grade. Conclusion DEGs and hub genes identified in the present study help us understand the molecular mechanisms of carcinogenesis and progression of glioma, and provide candidate targets for diagnosis and treatment of glioma.

Mining database for the clinical significance and prognostic value of ESRP1 in skin cutaneous melanoma

Background ESRP1 has been described as epithelium-specific splicing regulator involved in many tumors progression. However, the role of ESRP1 in skin cutaneous melanoma is unclear. In the present study, we explored the expression levels of ESRP1 in melanoma and its effect on prognosis and immune infiltration. Methods This study integrated the information from ONCOMINE, GEPIA and UALCAN databases. We explored the genes closely related to ESRP1 using LINKEDIMECS and further analyzed the target kinase, miRNA and transcription factors of ESRP1.GSEA software was used to analyze the changes of GO function enrichment and KEGG pathway after ESRP1 and its related 100 genes changes. Finally, TIMER was used to analyze the relationship between ESRP1 and tumor immune cell infiltration. Results The expression of ESRP1mRNA was significantly downregulated in SKCM patients in comparison with healthy control based on tumor stage and lymph node metastasis status. Low level of ESRP1 expression correlated with better overall survival. ESRP1 is specifically related to tumor-associated kinases CDK1, miRNA138, and transcription factors ETF. Functional network analysis demonstrated that ESRP1 is involved in the regulation of the condensed chromosome, epidermis development, translational initiation, ribosome, drug metabolism, and chemical carcinogenesis. Mechanistically, results suggested that ESRP1 might play an important role in regulating tumor-associated macrophages polarization, DCs infiltration, Treg cells and T cell exhaustion. Conclusion Our study demonstrates ESRP1 expression and its prognostic value and potential regulatory networks in SKCM, shedding light on the clinical significance of ESRP1 and providing a novel biomarker for determining prognosis and immune infiltration in SKCM.

Intranuclear DNA density affects chromosome condensation in metazoans

Chromosome condensation is critical for accurate inheritance of genetic information. The degree of condensation, which is reflected in the size of the condensed chromosomes during mitosis, is not constant. It is differentially regulated in embryonic and somatic cells. In addition to the developmentally programmed regulation of chromosome condensation, there may be adaptive regulation based on spatial parameters such as genomic length or cell size. We propose that chromosome condensation is affected by a spatial parameter called the chromosome amount per nuclear space, or “intranuclear DNA density.” Using Caenorhabditis elegans embryos, we show that condensed chromosome sizes vary during early embryogenesis. Of importance, changing DNA content to haploid or polyploid changes the condensed chromosome size, even at the same developmental stage. Condensed chromosome size correlates with interphase nuclear size. Finally, a reduction in nuclear size in a cell-free system from Xenopus laevis eggs resulted in reduced condensed chromosome sizes. These data support the hypothesis that intranuclear DNA density regulates chromosome condensation. This suggests an adaptive mode of chromosome condensation regulation in metazoans.

51 DEVELOPMENT OF EQUINE CLONED EMBRYOS DERIVED FROM IN VITRO-MATURED OOCYTES RECEIVING ADULT DERMAL FIBROBLAST CELL NUCLEI

The purpose of our study was to determine the in vitro developmental competences of equine NT embryos reconstructed with adult dermal fibroblast cells. Frozen/thawed fibroblast cells, whose mitotic cycle had been synchronized at G1/G0 stages through a contact inhibition of their migration and proliferative activity under total confluency, were used as a source of nuclear donor cells in the somatic cell cloning procedure. In vitro-matured oocytes were used as recipient cells for fibroblast cell nuclei. The compact cumulus–oocyte complexes (cpCOCs) were collected from abattoir-derived mare ovaries and selected for in vitro maturation. The cpCOCs were cultured in TC-199 medium supplemented with 5 mU mL–1 follicle-stimulating hormone (FSH), 10% fetal bovine serum (FBS) and 75 μg mL–1 kanamycin monosulfate (kanamycin A) for 30 h at 38.2°C in a 100% water-saturated atmosphere of 5% CO2 and 95% air. Cumulus-denuded in vitro-matured oocytes were incubated in the maturation medium supplemented with 0.4 μg mL–1 demecolcine for 40 min. The treated oocytes were subsequently transferred into TC-199 medium containing 4 mg mL–1 BSA-V and 5 μg mL–1 cytochalasin B. Metaphase chromosomes, which had been allocated into the chemically-induced protrusion of the plasma membrane, were removed microsurgically. The chemically-assisted enucleation was accomplished by gently aspirating the ooplasmic cone, which contained the condensed chromosome mass, with the aid of a beveled micropipette. The single nuclear donor cells were inserted into perivitelline space of previously enucleated oocytes. Fibroblast cell-ooplast couplets were fused with two consecutive DC pulses of 2.4 kV cm–1 for 30 μs. After a 1.5-h delay, nuclear transfer-derived oocytes were chemically activated by exposure to 5 μm L–1 calcium ionomycin for 5 to 7 min, followed by their incubation in B2 medium with addition of 2 mm L–1 6-dimethylaminopurine (6-DMAP) for 4 h. Reconstructed embryos were in vitro cultured in B2 medium for 2 days. Afterwards, cleaved embryos were co-cultured with Vero cells in B2 medium supplemented with 10% FBS for 5 to 6 days up to morula/blastocyst stages. From among 88 in vitro cultured cpCOCs, 55 (62.5%) acquired meiotic nuclear and cytoplasmic maturity state after reaching the Metaphase II stage. A total of 55 enucleated oocytes underwent reconstruction and 44/55 (80.0%) were successfully fused with nuclear donor cells. Out of 44 cultured NT embryos, 21 (47.7%) were cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages were 6/44 (13.6%) and 3/44 (6.8%), respectively. In conclusion, the cell nuclei of in vitro cultured adult dermal fibroblast cells, which had undergone the contact inhibition, were able to direct the preimplantation development of equine cloned embryos to morula and blastocyst stages. This work was supported by the Scientific Net of Animal Reproduction Biotechnology.