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Our view of the structure and function of the interphase nucleus has changed drastically in recent years. It is now widely accepted that the nucleus is a well organized and highly compartmentalized organelle and that this organization is intimately related to nuclear function. In this context, chromatin-initially considered a randomly entangled polymer-has also been shown to be structurally organized in interphase and its organization was found to be very important to gene regulation. Relevant and not completely answered questions are how chromatin organization is achieved and what mechanisms are responsible for changes in the positions of chromatin loci in the nucleus. A significant advance in the field resulted from tagging chromosome sites with bacterial operator sequences, and visualizing these tags using green fluorescent protein fused with the appropriate repressor protein. Simultaneously, fluorescence imaging techniques evolved significantly during recent years, allowing observation of the time evolution of processes in living specimens. In this context, the motion of the tagged locus was observed and analyzed to extract quantitative information regarding its dynamics. This review focuses on recent advances in our understanding of chromatin dynamics in interphase with the emphasis placed on the information obtained from single-particle tracking (SPT) experiments. We introduce the basis of SPT methods and trajectory analysis, and summarize what has been learnt by using this new technology in the context of chromatin dynamics. Finally, we briefly describe a method of SPT in a two-photon excitation microscope that has several advantages over methods based on conventional microscopy and review the information obtained using this novel approach to study chromatin dynamics. © 2008 Springer. Fil: Levi, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física. Laboratorio de Electrónica Cuántica; Argentina Fil: Gratton, Enrico. University of California at Irvine; Estados Unidos

The synthesis of lactose-operator gene fragments have been achieved by the improved triester methodology and new phosphorylating and condensing reagents.

The lac promoter maps between the represser i gene and the operator o gene, so that the operator gene is probably transcribed.

Phage 13vir is a sheath-tailed phage (Prozesky, de Klerk & Coetzee, 1965, Pl. 1, fig. 5) which arises from single clones of Proteus mirabilis strain 13 (Coetzee & Sacks, 1960). It forms large clear plaques on strain 13. The phage may carry a constitutive mutation in an operator gene which controls development of the prophage in strain 13. It is not inducible with ultraviolet light or mitomycin C but is often present in overnight broth cultures of strain 13. The phage is particularly troublesome in that strain 13 is the host of transducing phage 34 (Coetzee & Sacks, 1960) and lysates of the latter are usually contaminated with phage 13vir. When high titre φ 13vir. 13 (prepared by infecting a broth culture of strain 13) is spotted on a lawn of a locally isolated P. mirabilis strain 5006 the bacteria are killed but serial dilutions of the phage only sporadically form plaques.

The synthesis of lactose-operator gene fragments have been achieved by the improved triester methodology and new phosphorylating and condensing reagents.

Various methods of demonstrating the sites of plasma protein synthesis have been reported. Fundamentally, after the amino acids labelled with a radioactive isotope are added to a particular system and incubated for a certain period, then radioactivity is detected in particular proteins which increase by synthesis in the system studied. This procedure was first introduced by Peters and Anfinsen.110) That is, 14CO2 was added to the liver section of chickens, and albumin was separated after a certain time in which radioactive carbon was found to exist. Thus, it was proved that albumin is synthesized in the liver.

Background: Lactose repressor protein (Lac) controls the expression of the lactose metabolic genes in Escherichia coli by binding to an operator sequence in the promoter of the lac operon. Binding of inducer molecules to the Lac core domain induces changes in tertiary structure that are propagated to the DNA-binding domain through the connecting hinge region, thereby reducing the affinity for the operator. Protein–protein and protein–DNA interactions involving the hinge region play a crucial role in the allosteric changes occurring upon induction, but have not, as yet, been analyzed in atomic detail. Results: We have used nuclear magnetic resonance (NMR) spectroscopy and restrained molecular dynamics (rMD) to determine the structure of the Lac repressor DNA-binding domain (headpeice 62; HP62) in complex with a symmetrized lac operator. Analysis of the structures reveals specific interactions between Lac repressor and DNA that were not found in previously investigated Lac repressor–DNA complexes. Important differences with the previously reported structures of the HP56–DNA complex were found in the loop following the helix-turn-helix (HTH) motif. The protein–protein and protein–DNA interactions involving the hinge region and the deformations in the DNA structure could be delineated in atomic detail. The structures were also used for comparison with the available crystallographic data on the Lac and Pur repressor–DNA complexes. Conclusions: The structures of the HP62–DNA complex provide the basis for a better understanding of the specific recognition in the Lac repressor–operator complex. In addition, the structural features of the hinge region provide detailed insight into the protein–protein and protein–DNA interactions responsible for the high affinity of the repressor for operator DNA.