A conceptually novel role for Pax7 is found in zebrafish pigment formation. Absence of Pax7 leads to an expansion of the embryonic and larval melanophore lineage and a depletion of xanthophores, suggesting a model in which Pax7 is involved in early chromatophore specification processes.
There is an increasing interest in achieving gene regulation in biotechnological and biomedical applications by using synthetic DNA-binding agents. Most studies have so far focused on synthetic sequence-specific DNA-binding agents. Such approaches are relatively complicated and cost intensive and their level of sophistication is not always required, in particular for biotechnological application. Our study is inspired by in vivo data that suggest that DNA compaction might contribute to gene regulation. This study exploits the potential of using synthetic DNA compacting agents that are not sequence-specific to achieve gene regulation for in vitro systems. The semi-synthetic in vitro system we use include common cationic DNA-compacting agents, poly(amido amine) (PAMAM) dendrimers and the surfactant hexadecyltrimethylammonium bromide (CTAB), which we apply to linearized plasmid DNA encoding for the luciferase reporter gene. We show that complexing the DNA with either of the cationic agents leads to gene expression inhibition in a manner that depends on the extent of compaction. This is demonstrated by using a coupled in vitro transcription-translation system. We show that compaction can also protect DNA against degradation in a dose-dependent manner. Furthermore, our study shows that these effects are reversible and DNA can be released from the complexes. Release of DNA leads to restoration of gene expression and makes the DNA susceptible to degradation by Dnase. A highly charged polyelectrolyte, heparin, is needed to release DNA from dendrimers, while DNA complexed with CTAB dissociates with the non-ionic surfactant C12E5. Our results demonstrate the relation between DNA compaction by non-specific DNA-binding agents and gene expression and gene regulation can be achieved in vitro systems in a reliable dose-dependent and reversible manner.
We report that a 4.3 kbp linearised T7 DNA plasmid is actively transcribed when it is dispersed in the hexagonal liquid crystalline phase of dioleoylphosphoethanolamine (DOPE).
We used chicken retinospheroids (RS) to study the nuclear architecture of vertebrate cells in a three-dimensional (3D) cell culture system. The results showed that the different neuronal cell types of RS displayed an extreme form of radial nuclear organization. Chromatin was arranged into distinct radial zones which became already visible after DAPI staining. The distinct zones were enriched in different chromatin modifications and in different types of chromosomes. Active isoforms of RNA polymerase II were depleted in the outermost zone. Also chromocenters and nucleoli were radially aligned in the nuclear interior. The splicing factor SC35 was enriched at the central zone and did not show the typical speckled pattern of distribution. Evaluation of neuronal and non-neuronal chicken tissues showed that the highly ordered form of radial nuclear organization was also present in neuronal chicken tissues. Furthermore, the data revealed that the neuron-specific nuclear organization was remodeled when cells spread on a flat substrate. Monolayer cultures of a chicken cell line did not show this extreme form of radial organization. Rather, such monolayer cultures displayed features of nuclear organization which have been described before for many different types of monolayer cells. The finding that an extreme form radial nuclear organization, which has not been described before, is present in RS and tissues, but not in cells spread on a flat substrate, suggests that it would be important to complement studies on nuclear architecture performed with monolayer cells by studies on 3D cell culture systems and tissues.
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