Mutant differential labeling

The native CPMV virion could be reacted with the thiol reactive 5-maleimidofluoroscein, and it was found that 60 cysteine residues on the inside of each icosahcdral particle could be derivatized. A mutant form of CPMV was construeted with an exposed sulfhydryl on the exterior surface of the large subunit, and the resulting two cysteine residues could be differentially labeled, the exterior thiols reacting more rapidly than those on the interior. Qerivatization of thiol groups on the virus with a stilbene derivative for which antibodies were available showed the spatial differences between the location of thiols in the wild type and the mutant forms of the virus. The mutant, with thiols on the exterior of the virion, when derivatized with the stilbene, showed conjugate formation when reacted with the antibody. In contrast, attachment of the stilbene to the wild-type thiol on the interior of the virion showed no reactivity with the antibody. 

The methylation of dUMP by thymidylate synthetase is mediated by tetrahydrofolate and required for the formation of dTMP. Its loss by mutation creates a thymine-requiring auxotroph [127-129], Another alternate path for the synthesis of dTTP has been revealed by the use of the multimutational cytidine auxotrophs described above. Differential labeling of cytidine and uracil precursors in both E. coli [126] and Salmonella [125] shows that dTTP arises independently from either. Indeed, the contribution from cytidine is three to four times that from uracil. It is suggested that the second path involves a methylation of dCTP, then a deamination to give dTTP. One wonders why this pathway is not available in the thymine-requiring mutants which lack TMP synthetase. One explanation is that TMP synthetase may also function for the methylation of dCTP however, this has not yet been found to serve as an alternate substrate [130]. Two pathways may indeed exist in B. suhtilis, as indicated by the fact that two separate mutations are needed to obtain a thymine-requiring mutant [131]. 

With all separations there must be a tradeoff between yield and purity - high loss probabilities inevitably accompany high enrichment ratios. The underlying cause for this phenomenon is illustrated in Fig. 6.3. Although the mean mutant signal intensity can be maximally differentiated from wild type as discussed above, the expression, labeling and measurement processes introduce a point spread function that can cause... 

Fig. 3. Kinetic screening strategy. An initial library (106-107 complex) is incubated with biotinylated (or other suitable label) antigen (solid triangles) to saturate binding. The library is then incubated for a specified period of time in presence of excess unlabeled antigen (open triangles). FACS is used to visualize and isolate clones with the highest fluorescence having the slowest dissociation rate. The duration of the dissociation step (generally 3x - 5x the WT t1/2 calculated from Eq. 2) is adjusted to maximize the differentiation of the mutant(s) relative to the WT clone (see ref. 16 for greater detail).

The current test for the I1307K polymorphism uses allele-specific oligonucleotide hybridisation in which labelled probes are used to discriminate between wild type and mutant sequences. Such differential hybridisation requires considerable optimisation and is rarely perfect, resulting in a background signal that... 

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