The Number of Initiation Sites

Analysis of a second picornavirus was deemed important in light of the conflicting data from two laboratories, to demonstrate that the utilization of two initiation sites for vitro translation was not unique to a particular laboratory virus stock. Consequently, experiments similar to those described above were performed with protein-synthesizing extracts from LSc poliovirus-infected cells. The LSc strain is a multi-step, temperature-sensitive mutant derived from the Mahoney strain of poliovirus type 1. It was selected for these studies because previous work had suggested that initiation of translation of LSc virus proteins vivo showed a greater resistance to hypertonic salt treatment than did translation of Mahoney virus proteins (39)i and other workers have 

The results showed that translation of LSc virus ERA vitro also occurs at two different initiation sites, and the initiated tryptic peptides are identical with those produced by translation of Mhhoney virus ERA (41)  

A striking finding in these studies was that the relative rates of initiation at the two sites varied markedly as a function of the Mg concentration in the reaction mixture (59) Although the optimum Mg concentration for total fmet incorporation is approximately 2.0 mM, at Mg concentrations between 1.6 mM and 2.5 mM, initiation occurs predominantly at the site which generates tryptic peptide II hi er concentrations up to about 4 0 mM 

When the fmet-labeled polypeptides synthesized m vitro are analyzed directly by SDS-PAGE, two major bands are resolved (Knauert and Ehrenfeld, submitted). According to their mobilities in the gel, one has an apparent molecular weight of 115,000 daltons, whereas the second is a small polypeptide of approximately 6-9,000 daltons. Resolution of small polypeptides on these gels is poor, and the correlation between mobility and molecular weight in this range often breaks down. In addition, several minor bands are often 

Verification of the relationship between the polypeptides and the tryptic peptides was achieved by eluting the individual fmet-labeled proteins from a polyacrylamide gel and analyzing their trypsin digestion products. The high molecular weight polypeptide contains only tryptic peptide II, whereas the small polypeptide contains tiyptic peptide I. Three minor bands were individually analyzed in this way and all contained only tryptic peptide II. 

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Both the complexity and the sensitivity to initial states of the patterns generally increases as the number of initial sites with nonzero amplitude increases. As the number of nonzero sites increases, it also becomes increasingly likely that a change in the value of one of those sites (or a move in its location either to the left or right) will significantly alter the resulting pattern. 

The synthesis of complex polymer architectures by ATRP (or other living radical polymerizations) is useful but relatively restricted because the occurrence of bimolecular termination increases with the number of initiator sites on a polymeric species. 

In these experiments the number of initiating sites is a set value and cannot be increased. The rate of mutual destruction, the rate of diffusion of the monomer into the starch polymer, and the propagation rate are the controlling factors in the degree of grafting. The diffusion rate would be the critical factor governing the number of starch to acrylonitrile bonds achieved. These factors determine whether a block or a graft copolymer will be obtained. 

The formation of PAA star polymers using the core first method has been demonstrated in the ATRP process by use of multifunctional initiators [111, 112]. In this method, the number of arms in the star polymer can be determined by the number of initiating sites on the initiator. Star-shaped PtBuA was prepared by the arm first method via ATRP, using divinylbenzene, 1,4-butanediol diacrylate, and EGDMA as coupling reagents [113]. 

Subsequently, this concept has been extended to more highly functionalized star-initiators as well as polymeric initiators for the synthesis of graft structures. It should however, be appreciated that the polymerization process is still radical in nature and while radical—radical coupling reactions are decreased they are not eliminated and so as the number of initiating sites per molecule increase, the prob-... 

Inorganic macroinitiators can also be used for graft copolymerizations. Pendant vinyl functional pDMS was subjected to hydrosilation with 2-(4-chlorometh-ylphenyl)ethyldimethylsilane to prepare a multifunctional ATRP macroinitiator by Matyjaszewski et al. [233,234]. The ATRP of St was carried out using a pDMS macroinitiator with Mn=6600 and Mw/Mn=1.76 to yield a graft copolymer with Mn=14,800 and Mw/Mn=2.10. The increased polydispersity was attributed to the variation in the number of initiating sites on the pDMS backbone. NMR analysis showed that less than 5% of the total number of benzyl chloride moieties were left unreacted and that the weight ratio of pSt/pDMS was 1.18 [234]. 

Relative Initial Point Location Rule if the number of zero sites between nonzero sites is odd the resulting pattern is typically symmetrical and consists of melting ellipses if the number is even, the pattern is distorted or dies out [gross88bj. 

Further studies have shown that the degree of aggregation, and hence the number of active sites, is a function of the alkoxide substituent.813 For example, in (270) one of the i.vo-propoxide groups bridges two aluminum centers the other three are terminal ligands and all three initiate the ROP of CL. Less aggregated species such as (270) generally exhibit simple first order kinetics. 

Improved control was observed, however, upon addition of benzyl alcohol to the dinuclear complexes.887 X-ray crystallography revealed that whereas (296) simply binds the alcohol, (297) reacts to form a trinuclear species bearing four terminal alkoxides. The resultant cluster, (298), polymerizes rac-LA in a relatively controlled manner (Mw/Mn=1.15) up to 70% conversion thereafter GPC traces become bimodal as transesterification becomes increasingly prevalent. NMR spectroscopy demonstrates that the PLA bears BnO end-groups and the number of active sites was determined to be 2.5 0.2. When CL is initiated by (298) only 1.5 alkoxides are active and kinetic analysis suggests that the propagation mechanisms for the two monomers are different, the rate law being first order in LA, but zero order in CL. 

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