Protein Evolution: Mapping the Fitness Landscape and the Role of Constraints Imposed by the Genetic Code

Mutations are central to evolution, providing the genetic variation upon which selection acts. A mutation’s impact on fitness can be positive, negative, or neutral. Knowledge of the distribution of fitness effects (DFE) of mutations is fundamental for understanding evolutionary dynamics, molecular-level genetic variation, complex genetic disease, the accumulation of deleterious mutations, the molecular clock, and the impact of constraints imposed by the genetic code. In order to facilitate study of the DFE, we developed a new mutagenesis technique, termed PFunkel, by which large gene libraries can be created in a single day, single tube reaction with user-control over the type and position of mutations as well as the number of mutations per gene. We used PFunkel to create several types of libraries of the E. coli TEM-1 β-lactamase gene. By analyzing adaptive mutations in these libraries we found that the architecture of the genetic code significantly constrains the adaptive exploration of sequence space. However, the constraints endow the code with the ability to restrict access to amino acid mutations with a strong negative effect and, most remarkably, the ability to enrich for adaptive mutations. Furthermore, we present a comprehensive DFE for codon substitutions of the TEM-1 gene and amino acid substitutions in the TEM-1 protein. This DFE provides insight into the origin of the genetic code, support for the hypothesis that mRNA stability dictates codon usage at the beginning of genes, an extensive framework for understanding protein mutational tolerance, and evidence that mutational effects on protein thermodynamic stability shape the DFE. Contrary to prevailing expectations, we find that deleterious effects of mutation primarily arise from a decrease in specific protein activity and not protein cellular levels

Mammalian Inscuteable Regulates Spindle Orientation and Cell Fate in the Developing Retina

During mammalian neurogenesis, progenitor cells can divide with the mitotic spindle oriented parallel or perpendicular to the surface of the neuroepithelium. Perpendicular divisions are more likely to be asymmetric and generate one progenitor and one neuronal precursor. Whether the orientation of the mitotic spindle actually determines their asymmetric outcome is unclear. Here, we characterize a mammalian homolog of Inscuteable (mInsc), a key regulator of spindle orientation in Drosophila. mInsc is expressed temporally and spatially in a manner that suggests a role in orienting the mitotic spindle in the developing nervous system. Using retroviral RNAi in rat retinal explants, we show that downregulation of mInsc inhibits vertical divisions. This results in enhanced proliferation, consistent with a higher frequency of symmetric divisions generating two proliferating cells. Our results suggest that the orientation of neural progenitor divisions is important for cell fate specification in the retina and determines their symmetric or asymmetric outcome

Mammalian Inscuteable Regulates Spindle Orientation and Cell Fate in the Developing Retina

During mammalian neurogenesis, progenitor cells can divide with the mitotic spindle oriented parallel or perpendicular to the surface of the neuroepithelium. Perpendicular divisions are more likely to be asymmetric and generate one progenitor and one neuronal precursor. Whether the orientation of the mitotic spindle actually determines their asymmetric outcome is unclear. Here, we characterize a mammalian homolog of Inscuteable (mInsc), a key regulator of spindle orientation in Drosophila. mInsc is expressed temporally and spatially in a manner that suggests a role in orienting the mitotic spindle in the developing nervous system. Using retroviral RNAi in rat retinal explants, we show that downregulation of mInsc inhibits vertical divisions. This results in enhanced proliferation, consistent with a higher frequency of symmetric divisions generating two proliferating cells. Our results suggest that the orientation of neural progenitor divisions is important for cell fate specification in the retina and determines their symmetric or asymmetric outcome

Separase: a universal trigger for sister chromatid disjunction but not chromosome cycle progression

Separase is a protease whose liberation from its inhibitory chaperone Securin triggers sister chromatid disjunction at anaphase onset in yeast by cleaving cohesin's kleisin subunit. We have created conditional knockout alleles of the mouse Separase and Securin genes. Deletion of both copies of Separase but not Securin causes embryonic lethality. Loss of Securin reduces Separase activity because deletion of just one copy of the Separase gene is lethal to embryos lacking Securin. In embryonic fibroblasts, Separase depletion blocks sister chromatid separation but does not prevent other aspects of mitosis, cytokinesis, or chromosome replication. Thus, fibroblasts lacking Separase become highly polyploid. Hepatocytes stimulated to proliferate in vivo by hepatectomy also become unusually large and polyploid in the absence of Separase but are able to regenerate functional livers. Separase depletion in bone marrow causes aplasia and the presumed death of hematopoietic cells other than erythrocytes. Destruction of sister chromatid cohesion by Separase may be a universal feature of mitosis in eukaryotic cells

Cochlear progenitor number is controlled through mesenchymal FGF receptor signaling

The sensory and supporting cells (SCs) of the organ of Corti are derived from a limited number of progenitors. The mechanisms that regulate the number of sensory progenitors are not known. Here, we show that Fibroblast Growth Factors (FGF) 9 and 20, which are expressed in the non-sensory (Fgf9) and sensory (Fgf20) epithelium during otic development, regulate the number of cochlear progenitors. We further demonstrate that Fgf receptor (Fgfr) 1 signaling within the developing sensory epithelium is required for the differentiation of outer hair cells and SCs, while mesenchymal FGFRs regulate the size of the sensory progenitor population and the overall cochlear length. In addition, ectopic FGFR activation in mesenchyme was sufficient to increase sensory progenitor proliferation and cochlear length. These data define a feedback mechanism, originating from epithelial FGF ligands and mediated through periotic mesenchyme that controls the number of sensory progenitors and the length of the cochlea. DOI: http://dx.doi.org/10.7554/eLife.05921.00

Protein Evolution: Mapping the Fitness Landscape and the Role of Constraints Imposed by the Genetic Code

Mutations are central to evolution, providing the genetic variation upon which selection acts. A mutation’s impact on fitness can be positive, negative, or neutral. Knowledge of the distribution of fitness effects (DFE) of mutations is fundamental for understanding evolutionary dynamics, molecular-level genetic variation, complex genetic disease, the accumulation of deleterious mutations, the molecular clock, and the impact of constraints imposed by the genetic code. In order to facilitate study of the DFE, we developed a new mutagenesis technique, termed PFunkel, by which large gene libraries can be created in a single day, single tube reaction with user-control over the type and position of mutations as well as the number of mutations per gene. We used PFunkel to create several types of libraries of the E. coli TEM-1 β-lactamase gene. By analyzing adaptive mutations in these libraries we found that the architecture of the genetic code significantly constrains the adaptive exploration of sequence space. However, the constraints endow the code with the ability to restrict access to amino acid mutations with a strong negative effect and, most remarkably, the ability to enrich for adaptive mutations. Furthermore, we present a comprehensive DFE for codon substitutions of the TEM-1 gene and amino acid substitutions in the TEM-1 protein. This DFE provides insight into the origin of the genetic code, support for the hypothesis that mRNA stability dictates codon usage at the beginning of genes, an extensive framework for understanding protein mutational tolerance, and evidence that mutational effects on protein thermodynamic stability shape the DFE. Contrary to prevailing expectations, we find that deleterious effects of mutation primarily arise from a decrease in specific protein activity and not protein cellular levels