Norris Armstrong

CHAPTER 3 – Vectors for Phage Display

This chapter discusses various aspects of the vectors for phage display. A range of vectors is available for exogenous expression on the surface of bacteriophage M13 virus particles. The display sites most commonly used are within genes III or VIII, although there have been attempts at cloning in genes VII and IX. In several vectors, the displayed element is separated from the remainder of pill or pVIII, by short linkers. To reduce the level of nonrecombinants in phage display libraries, the reading frames of genes III or VIII have been intentionally destroyed in the stuffer sequence of some vectors. This has been accomplished by frame-shifting the gene when it was engineered with restriction sites. A mean for selecting for recombinant versus parental phage is to include a stop codon in the stuffer fragments of genes III or VIII. Under tetracycline selection, the fd-tet vector can be grown in bacterial cells as a plasmid, independent of phage production. This permits the propagation of engineered M 13 genomes which normally would not yield viable phage as plasmids inside bacteria. Suppressed stop codons have also been placed between displayed protein domains and domain II of pill. It is found that when such recombinants are propagated in a suppressor carrying a bacterial strain, virus particles are produced incorporating chimeric protein domain pill into the capsids. It is suggested that monovalent display can be a useful tool for the display of peptides and proteins that may not be well tolerated by M 13 in 5 or 2800 copies.

Identification and characterisation of Xenopus moesin, a Src substrate in Xenopus laevis oocytes.

The cortical actin cytoskeleton, consisting of actin filaments and actin binding proteins, immediately underlies the inner surface of the plasma membrane and is important both structurally and in relaying signals from the surface to the interior of the cell. Signal transduction processes, initiated in the cortex, modulate numerous cellular changes ranging from modifications of the local cytoskeleton structure, the position in the cell cycle, to cell behaviour. To examine the molecular mechanisms and events associated with cortical changes. We have investigated targets of the protein tyrosine kinase, Src, which is associated with the cortical cytoskeleton, in Xenopus laevis oocytes. When a mRNA encoding an activated form of Src tyrosine kinase (d-Src) is injected into oocytes several changes are observed: proteins are phosphorylated, the rate at which progesterone matures an oocyte to an egg is accelerated, and the cortex at the site of injection appears to contract. Previous studies have implicated actin filaments in the Src-stimulated cortical rearrangements. In this study we identify two actin binding proteins-cortactin and moesin--as Src substrates in Xenopus oocytes that are Src substrates. We cloned and characterised the cDNA encoding one of those, Xenopus moesin, a member of the ezrin/radixin/moesin (ERM) family of actin binding proteins. In addition, we have determined that moesin is recruited to the cortex at the site of Src mRNA injection.

Cell adhesion and cell signaling at gastrulation in the sea urchin

Abstract The sea urchin embryo follows a relatively simple cell behavioral sequence in its gastrulation movements. The embryo reaches the gastrula stage as a spherical monolayer of cells. To form the mesoderm, primary mesenchyme cells ingress by delaminating from the vegetal plate, crossing the basal lamina and moving into the central blastocoelar cavity. These cells then migrate along the basal lamina lining the blastocoel and eventually manufacture the skeleton. The presumptive secondary mesenchyme and endoderm invaginate as a tubular sheet of cells from the vegetal pole of the embryo. The archenteron extends across the blastocoel until its tip touches and attaches to the opposite side of the blastocoel. Secondary mesenchyme cells, originally at the tip of the archenteron, differentiate to form a variety of structures including coelomic pouches, esophageal muscles, pigment cells, and other cell types. The endoderm fuses with an invagination of the ventral ectoderm (the stomodaem), to form the mouth and complete the process of gastrulation. A number of experiments have established that these simple morphogenetic movements are accompanied by a number of cell adhesion changes plus a series of cell-cell interactions that provide spatial, temporal, and scalar information to cells of the mesoderm and endoderm. The requirement for cell signaling has been demonstrated by manipulative experiments where it has been shown that axial, temporal, spatial, and scalar information is obtained by mesoderm and endoderm from other embryonic cells. That information governs pattern formation and subsequent adhesive changes. This review describes the adhesion changes and the signaling that characterizes this early morphogenesis.