10K GENES PROJECT 2017-50

The 10K Genes Project is a BioBricks Foundation project aimed to synthesize and make freely available DNA in partnership with Twist Bioscience. Anyone around the world may request DNA to be synthesized through the 10K Genes Project as long as it is freely available to the scientific community. This document represents the week 50 of 2017 10K Genes Project order

Parameterization of a coarse-grained model of cholesterol with point-dipole electrostatics

© 2018, Springer Nature Switzerland AG. We present a new coarse-grained (CG) model of cholesterol (CHOL) for the electrostatic-based ELBA force field. A distinguishing feature of our CHOL model is that the electrostatics is modeled by an explicit point dipole which interacts through an ideal vacuum permittivity. The CHOL model parameters were optimized in a systematic fashion, reproducing the electrostatic and nonpolar partitioning free energies of CHOL in lipid/water mixtures predicted by full-detailed atomistic molecular dynamics simulations. The CHOL model has been validated by comparison to structural, dynamic and thermodynamic properties with experimental and atomistic simulation reference data. The simulation of binary DPPC/cholesterol mixtures covering the relevant biological content of CHOL in mammalian membranes is shown to correctly predict the main lipid behavior as observed experimentally

Creation and analysis of protein tyrosine phosphatase epsilon mutant mice

Addition and removal of phosphate from tyrosyl residues of proteins is an important mechanism for regulating the function and localization of proteins within the cell. Protein tyrosine phosphatases (PTP) are the family of enzymes that function to remove the phosphate specifically from phosphotyrosyl containing proteins. Protein tyrosine phosphatases act in concert with protein tyrosine kinases to provide intricate mechanisms for the regulation of cellular functions such as proliferation, differentiation, cell adhesion, and metabolism. This thesis revolves around the genetic analysis of protein tyrosine phosphatase-epsilon. This PTP exists as two isoforms - a transmembrane-containing receptor-like PTP having a small extracellular domain linked to two tandem phosphatase domains via a transmembrane segment, and an intracellular form containing a unique N-terminus attached to the two tandem phosphatase domains. The juxtamembrane region of the receptor-like form is common with the intracellular form and is defined as two exons in the mouse genome. Through the deletion of the transmembrane and first juxtamembrane exons of the Ptpre gene in mouse R1 embryonic stem cells using homologous recombination followed by Cre mediated excision of LoxP flanked exons we have generated mice mutant for these two forms of PTP-epsilon. The objectives of this thesis is to determine the functional role of the isoforms of protein tyrosine phosphatase epsilon (PTP-ε) through loss-of-function analysis in the mouse, and to test the hypothesis that murine PTP-ε is required for embryonic development and to determine if the gene product is required for reproduction. A further aim is to examine redundancy issues between the highly related protein tyrosine phosphatases, PTP-ε and RPTP-α. PtpreΔ/PtpreΔ mutant mice are viable and fertile. Gross anatomy and histological analysis of homozygous mutant mice shows no difference from wildtype. Protein tyrosine phosphatase-epsilon shows the strongest amino acid similarity to the receptor-like PTP-alpha. The high degree of similarity between these two molecules suggest that functional redundancy may occur. In collaboration with Dr. Jan Sap (NYU) we crossed the PtpraΔ/PtpraΔ mutant mouse to the PtpreΔ/PtpreΔ mutant mouse to create double knockout mice. Preliminary studies indicate that these mutant mice are viable, fertile and exhibit no apparent difference from wildtype with respect to behavior. Identification of subtle phenotypes in the laboratory context can be difficult, as important function of genes may not reveal themselves without the appropriate stresses or environment. This implies that a phenotype resulting from a particular gene knockout is present but may not be detectable by conventional methods.Medicine, Faculty ofMedical Genetics, Department ofGraduat