The organization of transcription in the nucleus of mammalian cells

Visions of the Cell Nucleus brings together scientists working on different aspects of structure and function of the mammalian cell nucleus. The goal of this book is to give a state-of-the-art overview on chromatin organization, function and dynamics of subnuclear structures, gene expression, DNA distribution, and disease. Emhpasis is put on new ideas of dynamic structure/function relationships revealed by the recent advent of new microscopy techniques as well as on subnuclear pathology and disease associations. The book provides all the information researchers, scientists and professionals need for this rapidly growing field

Fixation-induced redistribution of hyperphosphorylated RNA polymerase II in the nucleus of human cells

RNA polymerase II (pol II) transcribes the most varied group of genes and is present in hypo- and hyperphosphorylated forms, with residues Ser(2) and Ser(5) of the C-terminal domain (CTD) of the largest subunit as main targets of phosphorylation. The elongating (active) form is phosphorylated on Ser(2) and can be specifically recognized with the H5 antibody. It has been found in different nuclear distributions: in discrete sites throughout the nucleoplasm, consistent with a role in transcription, and/or concentrated in "splicing speckles", a nuclear compartment mostly devoid of transcriptional activity. Here, we assess the effects of cell fixation and permeabilization on the distribution of polymerase II and correlate its distribution with the preservation of cellular ultrastructure. We show that phospho-Ser(2) polymerase II can redistribute to, or be differentially retained in, "speckles" in conditions that do not preserve cellular ultrastructure. The fixation protocols that disrupt polymerase II distribution also cause partial or total loss of TATA-binding protein, Sm antigen and PML staining in PML bodies, and have no noticeable effect in the labeling of SC35 in "splicing speckles" or coilin in Cajal bodies. When nuclear ultrastructure is preserved, phospho-Ser(2) polymerase II is found in discrete sites throughout the nucleoplasm, without visible enrichment within splicing speckles. A minor proportion of the total amount of the phospho-Ser(2) form is present in these domains

Mice selected for differences in sensitivity to a benzodiazepine receptor inverse agonist vary in intermale aggression.

Brain y-aminobutyric acid (GABA) levels ars involved in intermale aggression in mice. It was therefore expected that animals genetically selected for their sensitivity to the convulsive effects of methyl B-carboline-3-carboxylate IB-CCM; BS, B-CCM sensitive, and BR, B-CCM resistant), a benzodiazepine (BZ) inverse agonist that specifically binds to the BZ site on the GABA-A receptor complex, would differ in their levels of aggressive behavior. Using two different aggression tests, in two independent experiments, we showed that BS mice are more aggressive than BR animals. The precise mechanisms underlying the observed line differences in B-CCM sensitivity and aggression remain to be determined

Trisomy 21 mid-trimester amniotic fluid induced pluripotent stem cells maintain genetic signatures during reprogramming: Implications for disease modeling and cryobanking

Trisomy 21 is the most common chromosomal abnormality and is associated primarily with cardiovascular, hematological, and neurological complications. A robust patient-derived cellular model is necessary to investigate the pathophysiology of the syndrome because current animal models are limited and access to tissues from affected individuals is ethically challenging. We aimed to derive induced pluripotent stem cells (iPSCs) from trisomy 21 human mid-trimester amniotic fluid stem cells (AFSCs) and describe their hematopoietic and neurological characteristics. Human AFSCs collected from women undergoing prenatal diagnosis were selected for c-KIT+and transduced with a Cre-lox-inducible polycistronic lentiviral vector encoding SOX2, OCT4, KLF-4, and c-MYC (50,000 cells at a multiplicity of infection (MOI) 1-5 for 72h). The embryonic stem cell (ESC)-like properties of the AFSC-derived iPSCs were established in vitro by embryoid body formation and in vivo by teratoma formation in RAG2-/-, γ-chain-/-, C2-/-immunodeficient mice. Reprogrammed cells retained their cytogenetic signatures and differentiated into specialized hematopoietic and neural precursors detected by morphological assessment, immunostaining, and RT-PCR. Additionally, the iPSCs expressed all pluripotency markers upon multiple rounds of freeze-thawing. These findings are important in establishing a patient-specific cellular platform of trisomy 21 to study the pathophysiology of the aneuploidy and for future drug discovery. © 2014 Mary Ann Liebert, Inc