Translocation methodology

The polymerase chain reaction is a methodology that is very well suited for the analysis of chromosomal translocations, particularly when the translocation breakpoints are clustered and the sequences flanking the translocation breakpoints are well characterized. 

The aim of this chapter is to describe the experimental approach and methodology which was utilized to isolate a large number of N-terminal isoforms of RARa, p, and /as well as to clone the PLZF-RARa cDNA from APL cells with t( 11 17) chromosomal translocation. It is worth pointing out that the same approach, with minor modifications, can be used to isolate N-terminal, or even C-terminal isoforms (55,56) of any gene for which some of the sequence is known. Therefore, this chapter could serve as a good reference for planning and executing such experiments. 

A methodology to generate alkenethiyl radical (2) starting from the easily available alkanethiyl radical (1) has recently been reported (Scheme 2) [28]. The reaction sequence consists of addition of opportunely substituted alkanethiyl radical (i.e. X = H, Y = Ph) to alkynes followed by 1,5-radical translocation and / -fragmentation. 

As translocation always involves a dynamic process, which cannot easily be studied by mere experimental techniques, above all, the application of long-term MD simulations should be implemented into the whole process of drug discovery and development. Due to significant increase in compnitational power and improvements in parallelization techniques, nowadays simulations of membrane transport proteins may stretch up to microseconds - that is, to physiologically relevant time scales. In this review we are describing the theory and methodology related to computational techniques used in the modeling of transporters and we will outline the recent developments in the field of ABC transporters and neurotransmitter transporters. 

It is obvious that the mechanism of action by which certain nutrients or drugs are translocated by a transporter implicates the protein to be flexible. In order to be able to allow for a sufficient comprehension of the dynamics of the transport protein, we can not only rely on experimental techniques. In addition, biomolecular simulations can provide a detailed description of particles in motion as a function of time. Thus, they are an important tool for understanding the physical basis of the structure and function of proteins, and biological macromolecules in general. However, experimental validation should always serve to test the accuracy of the calculated results and also to provide a basis for improving the methodology (Karplus McCammon, 2002). 

Recently, the field theory developed for neutral polymers has been extended to describe various polyelectrolytic systems in the absence/presence of externally added salt ions. The theory has been used to investigate the micro- and macrophase separation in polyelectrolyte systems [61-63], adsorption of polyelectrolytes on to the charged surfaces [64, 65], polyelectrolyte brushes [66, 67], confinement effects [14], counterion adsorption [15], translocation of polyelectrolytes (R. Kumar and M. Muthukumar, unpublished), and the assembly of single stranded RNA viruses (J. Wang, R. Kumar, and M. Muthukumar, unpublished). In this chapter, we review the general methodology behind the SCFT for polyelectrolytes. 

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