ProxiMAX randomisation:a new technology for non-degenerate saturation mutagenesis of contiguous codons

Back in 2003, we published ‘MAX’ randomisation, a process of non-degenerate saturation mutagenesis using exactly 20 codons (one for each amino acid) or else any required subset of those 20 codons. ‘MAX’ randomisation saturates codons located in isolated positions within a protein, as might be required in enzyme engineering, or else on one face of an alpha-helix, as in zinc finger engineering. Since that time, we have been asked for an equivalent process that can saturate multiple, contiguous codons in a non-degenerate manner. We have now developed ‘ProxiMAX’ randomisation, which does just that: generating DNA cassettes for saturation mutagenesis without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, ProxiMAX randomisation uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents. Thus it requires no specialised chemistry, reagents nor equipment and simply relies on a process of saturation cycling comprising ligation, amplification and digestion for each cycle. The process can encode both unbiased representation of selected amino acids or else encode them in pre-defined ratios. Each saturated position can be defined independently of the others. We demonstrate accurate saturation of up to 11 contiguous codons. As such, ProxiMAX randomisation is particularly relevant to antibody engineering

Milk losses and dynamics during perturbations in dairy cows differ with parity and lactation stage

Milk yield dynamics during perturbations reflect how cows respond to challenges. This study investigated the characteristics of 62,406 perturbations from 16,604 lactation curves of dairy cows milked with an automated milking system at 50 Belgian, Dutch, and English farms. The unperturbed lactation curve representing the theoretical milk yield dynamics was estimated with an iterative procedure fitting a model on the daily milk yield data that was not part of a perturbation. Perturbations were defined as periods of at least 5 d of negative residuals having at least 1 day that the total daily milk production was below 80% of the estimated unperturbed lactation curve. Every perturbation was characterized and split in a development and a recovery phase. Based hereon, we calculated both the characteristics of the perturbation as a whole, and the duration, slopes, and milk losses in the phases separately. A 2-way ANOVA followed by a pairwise comparison of group means was carried out to detect differences between these characteristics in different lactation stages (early, mid-early, mid-late, and late) and parities (first, second, and third or higher). On average, 3.8 +/- 1.9 (mean +/- standard deviation) perturbations were detected per lactation in the first 305 d after calving, corresponding to an estimated 92.1 +/- 135.8 kg of milk loss. Only 1% of the lactations had no perturbations. On average, 2.3 kg of milk was lost per day in the development phase, while the recovery phase corresponded to an average increase in milk production of 1.5 kg/d, and these phases lasted an average of 10.1 and 11.6 d, respectively. Perturbation characteristics were significantly different across parity and lactation stage groups, and early and mid-early perturbations in higher parities were found to be more severe with faster development rates, slower recovery rates, and higher milk losses. The method to characterize perturbations can be used for precision phenotyping purposes that look into the response of cows to challenges or that monitor applications (e.g., to evaluate the development and recovery of diseases and how these are affected by preventive actions or treatments)

High-Resolution Phenotypic Landscape of the RNA Polymerase II Trigger Loop

<div><p>The active sites of multisubunit RNA polymerases have a “trigger loop” (TL) that multitasks in substrate selection, catalysis, and translocation. To dissect the <i>Saccharomyces cerevisiae</i> RNA polymerase II TL at individual-residue resolution, we quantitatively phenotyped nearly all TL single variants <i>en masse</i>. Three mutant classes, revealed by phenotypes linked to transcription defects or various stresses, have distinct distributions among TL residues. We find that mutations disrupting an intra-TL hydrophobic pocket, proposed to provide a mechanism for substrate-triggered TL folding through destabilization of a catalytically inactive TL state, confer phenotypes consistent with pocket disruption and increased catalysis. Furthermore, allele-specific genetic interactions among TL and TL-proximal domain residues support the contribution of the funnel and bridge helices (BH) to TL dynamics. Our structural genetics approach incorporates structural and phenotypic data for high-resolution dissection of transcription mechanisms and their evolution, and is readily applicable to other essential yeast proteins.</p></div

Correction: High-Resolution Phenotypic Landscape of the RNA Polymerase II Trigger Loop

<p>Correction: High-Resolution Phenotypic Landscape of the RNA Polymerase II Trigger Loop</p

Plate phenotypes employed for the screening Pol II alleles <i>in vivo</i>.

<p>Plate phenotypes employed for the screening Pol II alleles <i>in vivo</i>.</p

Phenotypic analyses of evolutionary variants suggest context-dependent functions for many TL residues.

<p>(A) General growth fitness defects of the TL single-substituted variants observed in the TL across Pol I, II, III evolution including 38 Pol II, 42 Pol I and 42 Pol III amino acid variants relative to <i>Sce</i> Pol II. (B) Evolutionary TL variants in three mutant classes from the TL phenotypic landscape (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006321#pgen.1006321.g004" target="_blank">Fig 4A and 4B</a>). Existing variants from <i>Sce</i> Pol I are colored in blue, and existing variants from <i>Sce</i> Pol III are colored in red. <i>Sce</i> Pol I has three substitutions (V1089H, A1090G and S1091A) that cause LOF in the Pol II context; <i>Sce</i> Pol III has one substitution (A1076G) classified as GOF and one substitution (N1082K) classified as LOF. (C) Difference in positioning of funnel helices (relative to TL) in Pol I and Pol II. Cartoon representation of TL/funnel helices from Pol I and Pol II are shown in cyan and yellow, respectively (PDB: 5C4J and 2VUM).</p

Functional contribution of TL tip and Funnel Helix α-21 to proper TL dynamics.

<p>(A) Observed and predicted interactions between TL and TL-proximal domains. TL schematic is shown with residues identified by single-letter amino acid code and positions of interest annotated. Positions of GOF mutants isolated in our screen, along with the positions for a subset of previously isolated TL-proximal GOF mutants, are color coded in green. Observed TL interactions with other Rpb1 domains from structures or simulation studies are shown as grey dashed lines. (B) Maximal <i>in vitro</i> elongation rates (nucleotides/second) of Pol II WT and genetic GOF mutants S713P, I1327V and A1076T. (C) Observed interactions between open TL tip and TL adjacent charged residues (PDB: 5C4X). Funnel Helix refers to the Rpb1 α-21 alpha-helix. (D) Genetic interactions between the TL tip and proximal Rpb1 domains. Schematics of the TL and adjacent domains are shown in lines, with positions of interest shown in single-letter amino acid code. Substituted residues are shown in grey, with substituting amino acids shown in white, blue or green filled circles based on single substitution phenotypes (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006321#pgen.1006321.s010" target="_blank">S8F Fig</a>). Double substitution phenotypes are shown as colored lines connecting the two relevant single substitutions. Some sets of similar interactions were grouped into nodes to reduce complexity in interaction lines.</p