Cleaning up polymerase chain reactions: Artificially expanded genetic information systems (AEGIS) add extra nucleotide "letters" to DNA alphabets; oligonucleotides containing AEGIS nucleotides do not bind to natural DNA. This "orthogonality" is exploited here by placing two AEGIS nucleotides (P and Z) in external tags for primers targeting three cancer genes in a nested PCR architecture. AEGIS tags support multiplexed PCR with fewer primer dimers and off-target amplicons than multiplexed PCR without AEGIS components.
Experimental evolution via continuous culture is a powerful approach to the alteration of complex phenotypes, such as optimal/ maximal growth temperatures. The benefit of this approach is that phenotypic selection is tied to growth rate, allowing the production of optimized strains. Herein, we demonstrate the use of a recently described long-term culture apparatus called the Evolugator for the generation of a thermophilic descendant from a mesophilic ancestor (Escherichia coli MG1655). In addition, we used whole-genome sequencing of sequentially isolated strains throughout the thermal adaptation process to characterize the evolutionary history of the resultant genotype, identifying 31 genetic alterations that may contribute to thermotolerance, although some of these mutations may be adaptive for off-target environmental parameters, such as rich medium. We undertook preliminary phenotypic analysis of mutations identified in the glpF and fabA genes. Deletion of glpF in a mesophilic wild-type background conferred significantly improved growth rates in the 43-to-48°C temperature range and altered optimal growth temperature from 37°C to 43°C. In addition, transforming our evolved thermotolerant strain (EVG1064) with a wild-type allele of glpF reduced fitness at high temperatures. On the other hand, the mutation in fabA predictably increased the degree of saturation in membrane lipids, which is a known adaptation to elevated temperature. However, transforming EVG1064 with a wildtype fabA allele had only modest effects on fitness at intermediate temperatures. The Evolugator is fully automated and demonstrates the potential to accelerate the selection for complex traits by experimental evolution and significantly decrease development time for new industrial strains.
Standard procedures for using nuclear Overhauser enhancements (NOE) between protons to generate structures for diamagnetic proteins in solution from NMR data may be supplemented by using dipolar shifts if the protein is paramagnetic. This is advantageous since the electron -nuclear dipolar coupling provides relatively long-range geometric information with respect to the paramagnetic centre which complements the short-range distance constraints NOEs. Several different strategies have been developed to date, but none of these attempts to combine data from NOEs and dipolar shifts in the initial stages of structure calculation or to determine three dimensional protein structures together with their magnetic properties. This work shows that the magnetic and atomic structures are highly correlated and that it is important to have additional constraints both to provide starting parameters for the magnetic properties and to improve the definition of the best fit. Useful parameters can be obtained for haem proteins from Fermi contact shifts; this approach is compared with a new method based on the analysis of dipolar shifts in haem methyl groups with respect to data from horse and tuna ferricytochromes c. The methods developed for using data from NOEs and dipolar shifts have been incorporated in a new computer program, PARADYANA, which is demonstrated in application to a model data set for the sequence of the haem octapeptide known as microperoxidase-8.
Amphiregulin (AR), a heparin-binding, epidermal growth factor (EGF) receptor ligand has homology with EGF but exhibits a lower affinity for the EGF receptor than EGF. As the mature form of AR is truncated at the C terminus and lacks a conserved leucine residue known to be essential for high affinity binding of EGF to the EGF receptor, wild-type AR (AR1-84), a C-terminally extended AR construct incorporating six residues from the predicted coding sequence of AR (AR1-90) and a similarly extended construct with a Met86 to Leu substitution (AR1-90(leu86)) were expressed as recombinant proteins in yeast, purified by heparin affinity and C18 reverse phase chromatography and their relative biological activities determined. The growth factors were tested in mitogenesis and EGF receptor autophosphorylation assays and their relative order of potencies was found to be leu86> met86 > wt. The AR1-90(leu86) construct was found to be 50- to 100-fold more active than wild type AR1-84 consistent with previously reported studies of the role of the equivalent C-terminal leucine in EGF or TGF alpha. Significantly, the C-terminally extended form of AR, AR1-90, which utilized six residues from the predicted coding sequence, was 10-times more active than wild type AR1-84. This difference in activity of the C-terminally extended form of AR may be of biological significance since differential proteolytic processing of the AR precursor in vivo could result in production of multiple forms of the growth factor with differing affinities for the EGF receptor and hence differing biological potencies.
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