- ZNF384-fusion proteins have high affinity for the transcriptional coactivator EP300 and aberrant transcriptional activities. [Journal Article]
- FLFEBS Lett 2019 Jun 24
- Zinc-finger protein 384 (ZNF384) fusion (Z-fusion) genes have recently been identified as recurrent fusion genes in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and have been detected in 7…
Zinc-finger protein 384 (ZNF384) fusion (Z-fusion) genes have recently been identified as recurrent fusion genes in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and have been detected in 7-17% of Philadelphia chromosome-negative BCP-ALL cases. We selected SALL4 and ID2 as potential Z-fusion-specific transcriptional targets that might lead to the differentiation disorder of Z-fusion-positive ALL. The introduction of EP300-ZNF384 and SYNRG-ZNF384 induced the expression of these genes. Z-fusion proteins exhibited stronger transcriptional activities on the promoter or enhancer region of these genes than Wild-Z. Furthermore, GST pull-down assay revealed that Z-fusion proteins associated more strongly with EP300 than Wild-Z. Co-expression of EP300 specifically enhanced the transcriptional activities of Z-fusion proteins. We propose the increased EP300-binding of Z-fusion proteins as a mechanism for their increased transcriptional activities. This article is protected by copyright. All rights reserved.
- Chromosomal Localization of 18S-28S rDNA and (TTAGGG)n Sequences in Two South African Dormice of the Genus Graphiurus (Rodentia: Gliridae). [Journal Article]
- CGCytogenet Genome Res 2019 Jun 22
- Classical cytogenetics and mapping of 18S-28S rDNA and (TTAGGG)n sequences by fluorescence in situ hybridization (FISH) was performed on Graphiurus platyops (GPL) and Graphiurus ocularis (GOC) metaph…
Classical cytogenetics and mapping of 18S-28S rDNA and (TTAGGG)n sequences by fluorescence in situ hybridization (FISH) was performed on Graphiurus platyops (GPL) and Graphiurus ocularis (GOC) metaphases with the aim to characterize the genomes. In both species, inverted DAPI karyotypes showed the same diploid number, 2n = 46, and hybridization of the (TTAGGG)n probe revealed interstitial telomeric sequences (ITSs) at the centromeres of almost all bi-armed chromosomes. FISH with the rDNA probe localized nucleolus organizer regions (NORs), at the terminal ends of the p arms of the subtelocentric pairs 16 and 17 in both species and detected additional signals on GPL8 and GOC18, 19, and 22. The species have similar karyotypes, but their chromosome pairs 18-22 differ in morphology; these are acrocentric in G. platyops, as also confirmed by C-banding, and subtelocentric in G. ocularis. These differences in pairs 18-22 were also highlighted by hybridization of the telomeric probe (TTAGGG)n, which showed the small p arms in G. ocularis enriched with ITSs. FISH of rDNA probes detected multiple NOR loci in G. ocularis, underlining the intense evolutionary dynamics related to these genes. Although the Graphiurus species analyzed have similar karyotypes, the results on the repetitive sequences indicate a complex pattern of genomic reorganization and evolution occurring in these phylogenetically close species.
- Transcriptome analysis identified aberrant gene expression in pollen developmental pathways leading to CGMS in cotton (Gossypium hirsutum L.). [Journal Article]
- PlosPLoS One 2019; 14(6):e0218381
- Male sterility (induced or natural) is a potential tool for commercial hybrid seed production in different crops. Despite numerous endeavors to understand the physiological, hereditary, and molecular…
Male sterility (induced or natural) is a potential tool for commercial hybrid seed production in different crops. Despite numerous endeavors to understand the physiological, hereditary, and molecular cascade of events governing CMS in cotton, the exact biological process controlling sterility and fertility reconstruction remains obscure. During current study, RNA-Seq using Ion Torrent S5 platform is carried out to identify 'molecular portraits' in floral buds among the Cytoplasmic Genic Male Sterility (CGMS) line, its near-isogenic maintainer, and restorer lines. A total of 300, 438 and 455 genes were differentially expressed in CGMS, Maintainer, and Restorer lines respectively. The functional analysis using AgriGo revealed suppression in the pathways involved in biogenesis and metabolism of secondary metabolites which play an important role in pollen and anther maturation. Enrichment analysis showed dearth related to pollen and anther's development in sterile line, including anomalous expression of genes and transcription factors that have a role in the development of the reproductive organ, abnormal cytoskeleton formation, defects in cell wall formation. The current study found aberrant expression of DYT1, AMS and cytochrome P450 genes involved in tapetum formation, pollen development, pollen exine and anther cuticle formation associated to male sterility as well as fertility restoration of CGMS. In the current study, more numbers of DEGs were found on Chromosome D05 and A05 as compared to other chromosomes. Expression pattern analysis of fourteen randomly selected genes using qRT-PCR showed high concurrence with gene expression profile of RNA-Seq analysis accompanied by a strong correlation of 0.82. The present study provides an important support for future studies in identifying interaction between cyto-nuclear molecular portraits, to accelerate functional genomics and molecular breeding related to cytoplasmic male sterility studies in cotton.
- Clinical application of RNA sequencing in sarcoma diagnosis: An institutional experience. [Journal Article]
- MMedicine (Baltimore) 2019; 98(25):e16031
- Accurate diagnoses of sarcoma are sometimes challenging on conventional histomorphology and immunophenotype. Many specific genetic aberrations including chromosomal translocations have been identifie…
Accurate diagnoses of sarcoma are sometimes challenging on conventional histomorphology and immunophenotype. Many specific genetic aberrations including chromosomal translocations have been identified in various sarcomas, which can be detected by fluorescence in situ hybridization and polymerase chain reaction analysis. Next-generation sequencing-based RNA sequencing can screen multiple sarcoma-specific chromosome translocations/fusion genes in 1 test, which is especially useful for sarcoma without obvious differentiation. In this report, we utilized RNA sequencing on formalin-fixed paraffin-embedded (FFPE) specimens to investigate the possibility of diagnosing sarcomas by identifying disease-specific fusion genes. Targeted RNA sequencing was performed on 6 sarcoma cases. The expected genetic alterations (clear cell sarcoma/EWSR1-ATF1, Ewing sarcoma/EWSR1-FLI1, myxoid liposarcoma/DDIT3-FUS) in four cases were detected and confirmed by secondary tests. Interestingly, three SS18 fusion genes (SS18-SSX2B, SS18-SSX2, and SS18-SSX4) were identified in a synovial sarcoma case. A rare fusion gene (EWSR1-PATZ1) was identified in a morphologically challenging case; which enabled us to establish the diagnosis of low grade glioneural tumor. In conclusion, RNA sequencing on FFPE specimen is a reliable method in establishing the diagnosis of sarcoma in daily practice.
- Improved CUT&RUN chromatin profiling tools. [Journal Article]
- EElife 2019 Jun 24; 8
- Previously we described a novel alternative to Chromatin Immunoprecipitation, CUT&RUN, in which unfixed permeabilized cells are incubated with antibody, followed by binding of a Protein A-Micrococcal…
Previously we described a novel alternative to Chromatin Immunoprecipitation, CUT&RUN, in which unfixed permeabilized cells are incubated with antibody, followed by binding of a Protein A-Micrococcal Nuclease (pA/MNase) fusion protein (Skene and Henikoff, 2017). Here we introduce three enhancements to CUT&RUN: A hybrid Protein A-Protein G-MNase construct that expands antibody compatibility and simplifies purification, a modified digestion protocol that inhibits premature release of the nuclease-bound complex, and a calibration strategy based on carry-over of E. coli DNA introduced with the fusion protein. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling.
- Design of microaerobically inducible replicon for high-yield plasmid DNA production. [Journal Article]
- BBBiotechnol Bioeng 2019 Jun 24
- A pUC-derived replicon inducible by oxygen limitation was designed and tested in fed-batch cultures of Escherichia coli. It included the addition of a second inducible copy of rnaII, the positive rep…
A pUC-derived replicon inducible by oxygen limitation was designed and tested in fed-batch cultures of Escherichia coli. It included the addition of a second inducible copy of rnaII, the positive replication control element. The rnaII gene was expressed from Ptrc and cloned into pUC18 to test the hypothesis that the ratio of the positive control molecule RNAII to the negative control element, RNAI, was the determinant of plasmid copy number per chromosome (PCN). The construct was evaluated in several E. coli strains. Evaluations of the RNAII/RNAI ratio, PCN and plasmid yield normalized to biomass (YpDNA/X) were performed and the initial hypothesis was probed. Furthermore, in high cell-density cultures in shake flasks, an outstanding amount of 126 mg/L of plasmid was produced. The microaerobically inducible plasmid was obtained by cloning the rnaII gene under the control of the oxygen-responsive Vitreoscilla stercoraria hemoglobin promoter. For this plasmid, but not for pUC18, the RNAII/RNAI ratio, PCN and YpDNA/X efficiently increased after the shift to the microaerobic regime in fed-batch cultures in a 1 L bioreactor. The YpDNA/X of the inducible plasmid reached 12 mg/g at the end of the fed-batch but the original pUC18 only reached ca. 6 mg/g. The proposed plasmid is a valuable alternative for the operation and scale-up of plasmid DNA production processes in which mass transfer limitations will not represent an issue. This article is protected by copyright. All rights reserved.
- A case of de novo splice site variant in SLC35A2 showing developmental delays, spastic paraplegia, and delayed myelination. [Journal Article]
- MGMol Genet Genomic Med 2019 Jun 23; :e814
- CONCLUSIONS: This case would expand the phenotypic spectrum of SLC35A2-related disorders to delayed myelination with spasticity and no seizures.
- Mosaic paternal haploidy in a patient with pancreatoblastoma and Beckwith-Wiedemann spectrum. [Case Reports]
- AJAm J Med Genet A 2019 Jun 24
- Pancreatoblastoma is a rare type of pancreatic cancer in children. Here, we describe a case in which Beckwith-Wiedemann syndrome (BWS) was first suspected because of placental mesenchymal dysplasia. …
Pancreatoblastoma is a rare type of pancreatic cancer in children. Here, we describe a case in which Beckwith-Wiedemann syndrome (BWS) was first suspected because of placental mesenchymal dysplasia. Although the baby did not show the stigmata characteristic of BWS or abnormal peripheral blood methylation, she developed a massive pancreatoblastoma 2 months later. She survived after partial excision of the tumor and chemotherapy. The methylation pattern of the pancreatoblastoma tissue was typical of BWS. Single nucleotide polymorphism (SNP) array analyzes revealed that the pancreatoblastoma tissue had genome-wide loss of maternal alleles. Peripheral blood and nontumor pancreatic tissue showed normal biparental genomic contribution. Interphase fluorescence in situ hybridization analysis with centromeric probes for chromosomes 2 and 11 revealed haploid pancreatoblastoma cells, whereas the placental mesenchymal dysplasia tissue and nontumor pancreas tissue showed diploidy. SNP genotype analysis suggested the presence of mosaicism with the pancreatoblastoma tissue having a different paternal haplotype than that of the peripheral blood and nontumor pancreatic tissue. We report for the first time mosaic paternal haploidy associated with pancreatoblastoma. Babies with placental mesenchymal dysplasia, even those without a definitive diagnosis of BWS, need to be closely followed for the occurrence of embryonic tumors.
- Micro-bioreactor cultivations of Fab producing Escherichia coli reveal genome-integrated systems as suitable for prospective studies on direct Fab expression effects. [Journal Article]
- BJBiotechnol J 2019 Jun 24; :e1800637
- Despite efforts to develop concepts for efficient antibody fragment (Fab) production in Escherichia coli and the high degree of similarity within this protein class, we are far from a generic platfor…
Despite efforts to develop concepts for efficient antibody fragment (Fab) production in Escherichia coli and the high degree of similarity within this protein class, we are far from a generic platform technology. Indeed, feasible production of new Fab candidates remains challenging. In this study, we established a set-up that enables direct characterization of host cell response to Fab expression by utilizing genome-integrated systems. Among the multitude of factors that influence Fab expression, we varied the variable domain, the translocation mechanism, the host strain, as well as the copy number of the gene of interest. The resulting 32 production clones were characterized in carbon-limited micro-bioreactor cultivations with yields of 0 - 7.4 mg Fab/g cell dry mass. Antigen-binding region variations had the greatest effect on Fab yield. In most cases, the E. coli HMS174(DE3) strain performed better than the BL21(DE3) strain. Translocation mechanism variations mainly influenced leader peptide cleavage efficiency. Plasmid-free systems, with a single copy of the gene of interest integrated into the chromosome, reached Fab yields in the range of 80 to 300% of plasmid-based counterparts. Consequently, thegenome-integrated Fab production clones could greatly facilitate direct analyses of systems response to different impact factors under varying production conditions. This article is protected by copyright. All rights reserved.
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- Autism and social anxiety in children with sex chromosome trisomies: an observational study. [Journal Article]
- WOWellcome Open Res 2019; 4:32
- CONCLUSIONS: An increased risk of autism was found in girls with 47,XXX karyotype, as well as in boys with 47,XXY or 47,XYY. Symptoms of social anxiety were increased in all three karyotypes. There was wide variation in psychiatric status of children with the same karyotype, suggesting that an extra sex chromosome affects developmental stability in a non-specific way, with a diverse range of possible phenotypes.