Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition,bi-directionally from the primed protospacer

Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼350 new spacers acquired in priming events and identified a 5′-protospacer-GG-3′ protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2–3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.     

Molecular dynamics studies of the human CD4 protein

A dynamical model for an N-terminal fragment of the human CD4 protein has been determined by computer simulation. The protein has been studied both in vacuo and in solution. Data from both simulations agree moderately well with each other and with the crystal structure. All elements of secondary structure were retained during simulation. Point mutation and sequence replacement studies have shown that a loop in CD4, residues 40–52, is involved in binding with gp120, the human immunodeficiency virus surface glycoprotein.^1,2 Our results show that the gp120-binding loop and a few regions which bind to monoclonal antibodies and class II MHC molecules are the most highly motile areas of the protein. These results are consistent with the suggestion that CD4 binds to target molecules by using induced-fit contacts. © 1994 John Wiley & Sons, Inc.     

Lysine-derived cross-links in the egg shell membrane

Egg shell membrane protein contains significant quantities of the lysine-derived aldehyde, allysine, and its aldol condensation product. NaB³H₄ reduction followed by alkaline hydrolysis of purified protein revealed that there were six residues/1000 of both allysine and the reduced aldol while only traces of desmosine and isodesmosine were detected. The amino acid composition of the membrane protein did not resemble that of mammalian elastin.     

Free Edges in Epithelial Cell Sheets Stimulate Epidermal Growth Factor Receptor Signaling

The ability of epithelia to migrate and cover wounds is essential to maintaining their functions as physical barriers. Wounding induces many cues that may affect the transition to motility, including the immediate mechanical perturbation, release of material from broken cells, new interactions with adjacent extracellular matrix, and breakdown of physical separation of ligands from their receptors. Depending on the exact nature of wounds, some cues may be present only transiently or insignificantly. In many epithelia, activation of the epidermal growth factor receptor (EGFR) is a central event in induction of motility, and we find that its continuous activation is required for progression of healing of wounds in sheets of corneal epithelial cells. Here, we examine the hypothesis that edges, which are universally and continuously present in wounds, are a cue. Using a novel culture model we find that their presence is sufficient to cause activation of the EGFR and increased motility of cells in the absence of other cues. Edges that are bordered by agarose do not induce activation of the EGFR, indicating that activation is not due to loss of any specific type of cell–cell interaction but rather due to loss of physical constraints.     

Host-parasite associations of Eimeria spp. (Apicomplexa:Eimeriidae) in kangaroos and wallabies of the genus Macropus (Marsupialia:Macropodidae)

Faecal samples from 514 kangaroos and wallabies representing 12 species of the genus Macropus were examined for oocysts of Eimeria spp. Six species of Eimeria were redescribed from their type hosts, and on the basis of finding homologous oocysts in the faeces of other Macropus spp., host ranges for these coccidia were extended. Eimeria hestermani Mykytowycz, 1964 is redescribed from M. giganteus (eastern grey kangaroo) and is described from M. fuliginosus (western grey kangaroo), M. rufogriseus (red-necked wallaby), M. dorsalis (black-striped wallaby), and M. eugenii (tammar wallaby). E. toganmainensis Mykytowycz, 1964 is redescribed from M. rufus (red kangaroo) and the host range is extended to M. giganteus, M. fuliginosus, M. rufogriseus and M. eugenii. E. wilcanniensis Mykytowycz, 1964 is redescribed from M. rufus, and the host range is extended to M. giganteus, M. fuliginosus and M. robustus (euro or wallaroo). E. macropodis Wenyon & Scott, 1925 is redescribed from M. rufogriseus, and is described from M. giganteus, M. fuliginosus, M. rufus, M. irma (western brush wallaby), M. parryi (whip-tailed wallaby), M. dorsalis, M. eugenii, and M. parma (parma wallaby). E. fausti Yakimoff & Matschoulsky, 1936, E. cunnamullensis Mykytowycz, 1964 and E. purchasei Mykytowycz, 1964 are synonymized with E. macropodis. E. marsupialium Yakimoff & Matschoulsky, 1936 is redescribed from M. giganteus, and from M. fuliginosus. E. gungahlinensis Mykytowycz, 1964 is redescribed from M. fuliginosus, and from M. giganteus. Seven new species of Eimeria are described. E. flindersi, new species, is described from M. eugenii, M. rufogriseus, and M. antilopinus (antilopine wallaroo). E. prionotemni, new species, is described from M. eugenii, M. parryi, M. rufogriseus, M. agilis (agile wallaby) and M. dorsalis. E. mykytowyczi, new species, is described from M. agilis, M. antilopinus, and M. parryi. E. parryi, new species, is described from M. parryi. E. yathongensis, new species, is described from M. fuliginosus and M. giganteus. E. parma, new species, is described from M. parma, and E. desmaresti, new species, is described from M. rufogriseus. E. kogoni Mykytowycz, 1964, and E. rufusi Prasad, 1960 are considered species inquirendae. The host-parasite associations of these coccidia, and of similar species of Eimeria in other genera of Macropodoid marsupials, are discussed in relation to the postulated phylogeny of the hosts.     

Conformational and helicoidal analysis of the molecular dynamics of proteins: "curves," dials and windows for a 50 psec dynamic trajectory of BPTI

A new procedure for the graphic analysis of molecular dynamics (MD) simulations on proteins is introduced, in which comprehensive visualization of results and pattern recognition is greatly facilitated. The method involves determining the conformational and helicoidal parameters for each structure entering the analysis via the method "Curves," developed for proteins by Sklenar, Etchebest, and Lavery (Proteins: Structure, Function Genet. 6:46-60, 1989) followed by a novel computer graphic display of the results. The graphic display is organized systematically using conformation wheels ("dials") for each torsional parameter and "windows" on the range values assumed by the linear and angular helicoidal parameters, and is present in a form isomorphous with the primary structure per se. The complete time evolution of dynamic structure can then be depicted in a set of four composite figures. Dynamic aspects of secondary and tertiary structure are also provided. The procedure is illustrated with an analysis of a 50 psec in vacuo simulation on the 58 residue protein, bovine pancreatic trypsin inhibitor (BPTI), in the vicinity of the local minimum on the energy surface corresponding to a high resolution crystal structure. The time evolution of 272 conformational and 788 helicoidal parameters for BPTI is analyzed. A number of interesting features can be discerned in the analysis, including the dynamic range of conformational and helicoidal motions, the dynamic extent of 2 degrees structure motifs, and the calculated fluctuations in the helix axis. This approach is expected to be useful for a critical analysis of the effects of various assumptions about force field parameters, truncation of potentials, solvation, and electrostatic effects, and can thus contribute to the development of more reliable simulation protocols for proteins. Extensions of the analysis to present differential changes in conformational and helicoidal parameters is expected to be valuable in MD studies of protein complexes with substrates, inhibitors, and effectors and in determining the nature of structural changes in protein-protein interactions.