Expectations of duplicate gene retention under the gene duplicability hypothesis.

BACKGROUND: Gene duplication is an important process in evolution. What causes some genes to be retained after duplication and others to be lost is a process not well understood. The most prevalent theory is the gene duplicability hypothesis, that something about the function and number of interacting partners (number of subunits of protein complex, etc.), determines whether copies have more opportunity to be retained for long evolutionary periods. Some genes are also more susceptible to dosage balance effects following WGD events, making them more likely to be retained for longer periods of time. One would expect these processes that affect the retention of duplicate copies to affect the conditional probability ratio after consecutive whole genome duplication events. The probability that a gene will be retained after a second whole genome duplication event (WGD2), given that it was retained after the first whole genome duplication event (WGD1) versus the probability a gene will be retained after WGD2, given it was lost after WGD1 defines the probability ratio that is calculated. RESULTS: Since duplicate gene retention is a time heterogeneous process, the time between the events (t1) and the time since the most recent event (t2) are relevant factors in calculating the expectation for observation in any genome. Here, we use a survival analysis framework to predict the probability ratio for genomes with different values of t1 and t2 under the gene duplicability hypothesis, that some genes are more susceptible to selectable functional shifts, some more susceptible to dosage compensation, and others only drifting. We also predict the probability ratio with different values of t1 and t2 under the mutational opportunity hypothesis, that probability of retention for certain genes changes in subsequent events depending upon how they were previously retained. These models are nested such that the mutational opportunity model encompasses the gene duplicability model with shifting duplicability over time. Here we present a formalization of the gene duplicability and mutational opportunity hypotheses to characterize evolutionary dynamics and explanatory power in a recently developed statistical framework. CONCLUSIONS: This work presents expectations of the gene duplicability and mutational opportunity hypotheses over time under different sets of assumptions. This expectation will enable formal testing of processes leading to duplicate gene retention.

Dosage balance acts as a time-dependent selective barrier to subfunctionalization.

BACKGROUND: Gene duplication is an important process for genome expansion, sometimes allowing for new gene functions to develop. Duplicate genes can be retained through multiple processes, either for intermediate periods of time through processes such as dosage balance, or over extended periods of time through processes such as subfunctionalization and neofunctionalization. RESULTS: Here, we built upon an existing subfunctionalization Markov model by incorporating dosage balance to describe the interplay between subfunctionalization and dosage balance to explore selective pressures on duplicate copies. Our model incorporates dosage balance using a biophysical framework that penalizes the fitness of genetic states with stoichiometrically imbalanced proteins. These imbalanced states cause increased concentrations of exposed hydrophobic surface areas, which cause deleterious mis-interactions. We draw comparison between our Subfunctionalization + Dosage-Balance Model (Sub + Dos) and the previous Subfunctionalization-Only (Sub-Only) Model. This comparison includes how the retention probabilities change over time, dependent upon the effective population size and the selective cost associated with spurious interaction of dosage-imbalanced partners. We show comparison between Sub-Only and Sub + Dos models for both whole-genome duplication and small-scale duplication events. CONCLUSION: These comparisons show that following whole-genome duplication, dosage balance serves as a time-dependent selective barrier to the subfunctionalization process, by causing an overall delay but ultimately leading to a larger portion of the genome retained through subfunctionalization. This higher percentage of the genome that is ultimately retained is caused by the alternative competing process, nonfunctionalization, being selectively blocked to a greater extent. In small-scale duplication, the reverse pattern is seen, where dosage balance drives faster rates of subfunctionalization, but ultimately leads to a smaller portion of the genome retained as duplicates. This faster rate of subfunctionalization is because the dosage balance of interacting gene products is negatively affected immediately after duplication and the loss of a duplicate restores the stoichiometric balance. Our findings provide support that the subfunctionalization of genes that are susceptible to dosage balance effects, such as proteins involved in complexes, is not a purely neutral process. With stronger selection against stoichiometrically imbalanced gene partners, the rates of subfunctionalization and nonfunctionalization slow; however, this ultimately leads to a greater proportion of subfunctionalized gene pairs.

Models for the retention of duplicate genes and their biological underpinnings.

Gene content in genomes changes through several different processes, with gene duplication being an important contributor to such changes. Gene duplication occurs over a range of scales from individual genes to whole genomes, and the dynamics of this process can be context dependent. Still, there are rules by which genes are retained or lost from genomes after duplication, and probabilistic modeling has enabled characterization of these rules, including their context-dependence. Here, we describe the biology and corresponding mathematical models that are used to understand duplicate gene retention and its contribution to the set of biochemical functions encoded in a genome.

WGDTree: a phylogenetic software tool to examine conditional probabilities of retention following whole genome duplication events.

BACKGROUND: Multiple processes impact the probability of retention of individual genes following whole genome duplication (WGD) events. In analyzing two consecutive whole genome duplication events that occurred in the lineage leading to Atlantic salmon, a new phylogenetic statistical analysis was developed to examine the contingency of retention in one event based upon retention in a previous event. This analysis is intended to evaluate mechanisms of duplicate gene retention and to provide software to generate the test statistic for any genome with pairs of WGDs in its history. RESULTS: Here a software package written in Python, 'WGDTree' for the analysis of duplicate gene retention following whole genome duplication events is presented. Using gene tree-species tree reconciliation to label gene duplicate nodes and differentiate between WGD and SSD duplicates, the tool calculates a statistic based upon the conditional probability of a gene duplicate being retained after a second whole genome duplication dependent upon the retention status after the first event. The package also contains methods for the simulation of gene trees with WGD events. After running simulations, the accuracy of the placement of events has been determined to be high. The conditional probability statistic has been calculated for Phalaenopsis equestris on a monocot species tree with a pair of consecutive WGD events on its lineage, showing the applicability of the method. CONCLUSIONS: A new software tool has been created for the analysis of duplicate genes in examination of retention mechanisms. The software tool has been made available on the Python package index and the source code can be found on GitHub here: https://github.com/cnickh/wgdtree .

Fewer Exposed Lysine Residues May Explain Relative Resistance of Chicken Serum Albumin to In Vitro Protein Glycation in Comparison to Bovine Serum Albumin.

Protein glycation and formation of advanced glycation end products is associated with several diseases resulting from high blood glucose concentrations. Plasma albumin is directly exposed to circulating glucose concentrations and is therefore at greater risk of glycation than hemoglobin. As plasma glucose concentrations in birds are 1.5-2 times higher than mammals of similar mass, avian albumin may be particularly at risk of glycation. Thus, the goal of the present study was to compare the in vitro formation of glycated albumin in chicken serum albumin (CSA) and bovine serum albumin (BSA) exposed to a range of glucose concentrations over a 16-week period. The level of glycation for CSA and BSA was quantified using boronate affinity columns to separate glycated albumin from non-glycated albumin and calculating the difference in protein concentration of each sample. The results indicate that CSA is glycated to a lesser degree than BSA when the albumins are exposed to increasing concentrations of glucose (38.8-500 mM). This was most apparent at week sixteen (500 mM glucose) where BSA expressed a higher degree of glycation (37.8 ± 0.76%) compared to CSA (19.7 ± 1.06%, P < 0.05). Additionally, percent glycation at week sixteen was significantly higher than the glucose-free solutions for both BSA and CSA, indicating that glycation is glucose-dependent. Analyses of the protein structures suggest that the relative resistance of CSA to glycation may be due to fewer lysine residues and variations in protein folding that shield more lysine residues from the plasma. Moreover, comparisons of reconstructed ancestral albumin sequences show that the ancestor of birds had 6-8 fewer lysine residues compared to that of mammals.

Evolutionary Processes and Biophysical Mechanisms: Revisiting Why Evolved Proteins Are Marginally Stable.

Evolved proteins observed in natural organisms are found to be only marginally stable. Several mechanistic hypotheses have been presented to date to explain this observation. One idea that has been put forward is that active selection prevents proteins from becoming too stable to enable proper function. A second idea is that marginal stability reflects the point of mutation-selection-drift balance, where it is mutational pressure that generates marginal stability. A third idea explored in this issue of Journal of Molecular Evolution is that a physical limit prevents the evolution of more stable proteins rather than an evolutionary process. While the first two notions are based upon specific evolutionary processes, discussion here is aimed at reconciling evolutionary processes with the physics of protein folding, drawing upon the ideas that have been presented.

Proteomic analysis and immunogenicity of Mannheimia haemolytica vesicles.

Mannheimia haemolytica, a major causative agent in bovine respiratory disease, inflicts extensive losses each year on cattle producers. Commercially available vaccines are only partially efficacious. Immunity to M. haemolytica requires antibodies to secreted toxins and outer membrane proteins (OMPs) of the bacterium. Gram-negative bacteria produce membrane blebs or vesicles, the membrane components of which are primarily derived from OMPs. Accordingly, vesicles have been used as immunogens with various degrees of success. This study characterized components of M. haemolytica vesicles and determined their immunogenicity in mice and cattle. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of vesicles from this bacterium identified 226 proteins, of which 58 (25.6%) were OMPs and periplasmic and one (0.44%) was extracellular. Vesicles were used to vaccinate dairy calves and BALB/c mice. Analyses of sera from calves and mice by enzyme-linked immunosorbent assay (ELISA) showed that circulating antibodies against M. haemolytica whole cells and leukotoxin were significantly higher on days 21 and 28 (P < 0.05) than on day 0. For control calves and mice, there were no significant differences in serum anti-whole-cell and leukotoxin antibody levels from days 0 and 21 or 28, respectively. Lesion scores of lungs from vaccinated calves (15.95%) were significantly (P < 0.05) lower than those from nonvaccinated calves (42.65%). Sera from mice on day 28 and calves on day 21 showed 100% serum bactericidal activity. Sera from vesicle-vaccinated mice neutralized leukotoxin.

Immunogenicity of Mannheimia haemolytica recombinant outer membrane proteins serotype 1-specific antigen, OmpA, OmpP2, and OmpD15.

We previously identified Mannheimia haemolytica outer membrane proteins (OMPs) that may be important immunogens by using immunoproteomic analyses. Genes for serotype 1-specific antigen (SSA-1), OmpA, OmpP2, and OmpD15 were cloned and expressed, and recombinant proteins were purified. Objective 1 of this study was to demonstrate immunogenicity of the four recombinant OMPs in mice and cattle. Objective 2 was to determine if the addition of individual recombinant OMPs or combinations of them would modify immune responsiveness of mice to the recombinant chimeric protein SAC89, containing the main epitope from M. haemolytica outer membrane lipoprotein PlpE and the neutralizing epitope of M. haemolytica leukotoxin. Mice vaccinated with recombinant OmpA (rOmpA), rSSA-1, rOmpD15, and rOmpP2 developed significant antibody responses to M. haemolytica outer membranes and to the homologous recombinant OMP. Cattle vaccinated with rOmpA and rSSA-1 developed significant antibodies to M. haemolytica outer membranes by day 28, whereas cattle vaccinated with rOmpD15 and rOmpP2 developed only minimal responses. Sera from cattle vaccinated with each of the recombinant proteins stimulated complement-mediated killing of the bacterium. Concurrent vaccination with SAC89 plus any of the four rOMPs singly resulted in increased endpoint anti-SAC89 titers, and for the SAC89/rSSA-1 vaccinees, the response was increased significantly. In contrast, the SAC89/P2/SSA-1 and SAC89/OmpA/P2/D15/SSA-1 combination vaccines resulted in significant decreases in anti-SAC89 antibodies compared to SAC89 vaccination alone. In conclusion, under the conditions of these experiments, vaccination of mice and cattle with rOmpA and rSSA-1 stimulated high antibody responses and may have protective vaccine potential.

Identification and immunogenicity of Mannheimia haemolytica S1 outer membrane lipoprotein PlpF.

Immunity against Mannheimia haemolytica requires antibodies against leukotoxin (LKT) and bacterial cell surface antigens, most likely immunogenic outer membrane proteins (OMPs). Five immunogenic outer membrane lipoproteins identified and characterized in M. haemolytica were designated Pasteurella lipoproteins (Plp) A, -B, -C, -D and -E. Using immunoproteomics, we identified a heretofore-uncharacterized M. haemolytica immunogenic outer membrane lipoprotein that we designated PlpF, which was previously designated in the published sequence as a conserved hypothetical protein. We cloned and expressed rPlpF from two M. haemolytica serotype 1 strains (SAC159 and SAC160) and demonstrated a variable number of perfect (KKTEED) or imperfect (KKaEEa) repeats between residues 41 and 76 on the N-terminus. Antigenicity plots predicted the N-terminus repeat region to be highly antigenic. The plpF gene in multiple M. haemolytica S1, S2, and S6 isolates varied in the number of repeats from three to seven. C-terminal region was highly conserved. Immunization of mice with SAC159 or SAC160 demonstrated immunogenicity in a dose-response manner. Immunization of calves demonstrated an increase in antibodies to PlpF, and rPlpF antibodies stimulated complement-mediated killing of M. haemolytica. Because calves had pre-existing anti-M. haemolytica antibodies due to prior natural exposure, functionality of the anti-PlpF antibody responses were demonstrated by marked reduction of complement-mediated killing by blocking of anti-PlpF antibodies with rPlpF In conclusion, PlpF might have vaccination potential against M. haemolytica infection in cattle.