Using defined finger–finger interfaces as units of assembly for constructing zinc-finger nucleases

Zinc-finger nucleases (ZFNs) have been used for genome engineering in a wide variety of organisms; however, it remains challenging to design effective ZFNs for many genomic sequences using publicly available zinc-finger modules. This limitation is in part because of potential finger–finger incompatibility generated on assembly of modules into zinc-finger arrays (ZFAs). Herein, we describe the validation of a new set of two-finger modules that can be used for building ZFAs via conventional assembly methods or a new strategy—finger stitching—that increases the diversity of genomic sequences targetable by ZFNs. Instead of assembling ZFAs based on units of the zinc-finger structural domain, our finger stitching method uses units that span the finger–finger interface to ensure compatibility of neighbouring recognition helices. We tested this approach by generating and characterizing eight ZFAs, and we found their DNA-binding specificities reflected the specificities of the component modules used in their construction. Four pairs of ZFNs incorporating these ZFAs generated targeted lesions in vivo, demonstrating that stitching yields ZFAs with robust recognition properties.     

Toward the structure of presenilin/γ-secretase and presenilin homologs

Presenilin is the catalytic component of the γ-secretase complex, a membrane-embedded aspartyl protease that plays a central role in biology and in the pathogenesis of Alzheimer’s disease. Upon assembly with its three protein cofactors (nicastrin, Aph-1 and Pen-2), presenilin undergoes autoproteolysis into two subunits, each of which contributes one of the catalytic aspartates to the active site. A family of presenilin homologs, including signal peptide peptidase, possess proteolytic activity without the need for other protein factors, and these simpler intramembane aspartyl proteases have given insight into the action of presenilin within the γ-secretase complex. Cellular and molecular studies support a nine-transmembrane topology for presenilins and their homologs, and small-molecule inhibitors and cysteine scanning with crosslinking have suggested certain presenilin residues and regions that contribute to substrate recognition and handling. Identification of partial complexes has also offered clues to protein–protein interactions within the γ-secretase complex. Biophysical methods have allowed 3D views of the γ-secretase complex and presenilins. Most recently, the crystal structure of a microbial presenilin homolog has confirmed a nine-transmembrane topology and intramembranous location and proximity of the two conserved and essential aspartates. The crystal structure also provides a platform for the formulation of specific hypotheses regarding substrate interaction and catalysis as well as the pathogenic mechanism of Alzheimer-causing presenilin mutations. This article is part of a Special Issue entitled: Intramembrane Proteases.     

Vitalism and the Resistance to Experimentation on Life in the Eighteenth Century

There is a familiar opposition between a ‘Scientific Revolution’ ethos and practice of experimentation, including experimentation on life, and a ‘vitalist’ reaction to this outlook. The former is often allied with different forms of mechanism – if all of Nature obeys mechanical laws, including living bodies, ‘iatromechanism’ should encounter no obstructions in investigating the particularities of animal-machines – or with more chimiatric theories of life and matter, as in the ‘Oxford Physiologists’. The latter reaction also comes in different, perhaps irreducibly heterogeneous forms, ranging from metaphysical and ethical objections to the destruction of life, as in Margaret Cavendish, to more epistemological objections against the usage of instruments, the ‘anatomical’ outlook and experimentation, e.g. in Locke and Sydenham. But I will mainly focus on a third anti-interventionist argument, which I call ‘vitalist’ since it is often articulated in the writings of the so-called Montpellier Vitalists, including their medical articles for the Encyclopédie. The vitalist argument against experimentation on life is subtly different from the metaphysical, ethical and epistemological arguments, although at times it may borrow from any of them. It expresses a Hippocratic sensibility – understood as an artifact of early modernity, not as some atemporal trait of medical thought – in which Life resists the experimenter, or conversely, for the experimenter to grasp something about Life, it will have to be without torturing or radically intervening in it. I suggest that this view does not have to imply that Nature is something mysterious or sacred; nor does the vitalist have to attack experimentation on life in the name of some ‘vital force’ – which makes it less surprising to find a vivisectionist like Claude Bernard sounding so close to the vitalists.     

The Hsp70/90 cochaperone,Sti1, suppresses proteotoxicity by regulating spatial quality control of amyloid-like proteins

Conformational diseases are associated with the conversion of normal proteins into aggregation-prone toxic conformers with structures similar to that of β-amyloid. Spatial distribution of amyloid-like proteins into intracellular quality control centers can be beneficial, but cellular mechanisms for protective aggregation remain unclear. We used a high-copy suppressor screen in yeast to identify roles for the Hsp70 system in spatial organization of toxic polyglutamine-expanded Huntingtin (Huntingtin with 103Q glutamine stretch [Htt103Q]) into benign assemblies. Under toxic conditions, Htt103Q accumulates in unassembled states and speckled cytosolic foci. Subtle modulation of Sti1 activity reciprocally affects Htt toxicity and the packaging of Htt103Q into foci. Loss of Sti1 exacerbates Htt toxicity and hinders foci formation, whereas elevation of Sti1 suppresses Htt toxicity while organizing small Htt103Q foci into larger assemblies. Sti1 also suppresses cytotoxicity of the glutamine-rich yeast prion [RNQ+] while reorganizing speckled Rnq1–monomeric red fluorescent protein into distinct foci. Sti1-inducible foci are perinuclear and contain proteins that are bound by the amyloid indicator dye thioflavin-T. Sti1 is an Hsp70 cochaperone that regulates the spatial organization of amyloid-like proteins in the cytosol and thereby buffers proteotoxicity caused by amyloid-like proteins.     

G1 arrest and the division/conjugation decision in Tetrahymena.

The transformation from the asexual proliferative stage of Tetrahymena to the sexual stage, during which cells of complementary mating types pair and nuclear fertilization occurs, provides an opportunity to study the relationship between the division cycle and differentiation. Conjugation is induced in cells starved for at least 2 hr by mixing complementary mating types. To determine the effect of starvation on the cell cycle, dividing cells were selected from a log growth culture and stepped down to non-nutrient conditions. The G₁ stage is operationally divisible into two sectors, A and B. In the A stage, cells arrest in nutrient-free medium. In the B stage, they proceed through the division cycle. Arrested G₁A cells may conjugate directly when challenged with similar cells of a complementary mating type. It is thereby demonstrated that Tetrahymena cells in G₁A can be directed to divide (nutrient conditions) or can be directed to differentiate (non-nutrient conditions plus complementary mating type) without an intervening division cycle. This rules out a requirement for reprogramming via chromosomal replication or cell division and suggests that G₁A is a stage during which the division/differentiation decision is made in direct response to ambient conditions.     

A cytological study of micronuclear elongation during conjugation in Tetrahymena

Micronuclear elongation is the first major event in a series of nuclear changes occurring during the sexual stage of the life cycle of Tetrahymena. Beginning at about one hour after cells of complementary mating types have conjugated, the micronucleus leaves its recess in the macronucleus and swells slightly. This is accompanied by a reorganization of its chromatin from a reticular to a solid body. In the next stage the micronucleus assumes an egg shape, a development concomitant with the appearance of microtubules. While the chromatin spins out from the dense body, and microtubules increase in number, the nucleus assumes a spindle shape. During the elongation, which increases the length of the nucleus some fifty fold, microtubules are prominent in clusters just internal to the nuclear membrane, and parallel to the longitudinal axis of the nucleus. When elongation is completed the nucleus is curved around the macronucleus. Internally, partially condensed strands of chromatin are located off-center, towards the macronuclear side, and the density of the microtubules is diminished. At all the stages, DNA is located throughout the nucleus; neither discrete chromosomes nor synaptonemal complexes are seen. Occasionally cytoplasmic membrane systems are seen fused to the nuclear envelope which retains the typical appearance of a double membrane with pores.     

Cytoplasmic filaments and cellular wound healing in amoeba proteus

The flexibility and self-healing properties of animal cell surface membranes are well known. These properties have been best exploited in various micrurgical studies on living cells (2, 3), especially in amoebae (7, 20). During nuclear transplantation in amoebae, the hole in the membrane through which a nucleus passes can have a diameter of 20-30 μm, and yet such holes are quickly sealed, although some cytoplasm usually escapes during the transfer. While enucleating amoebae in previous studies, we found that if a very small portion of a nucleus was pushed through the membrane and exposed to the external medium, the amoeba expelled such a nucleus on its own accord. When this happened, a new membrane appeared to form around the embedded portion of the nucleus and no visible loss of cytoplasm occurred during nuclear extrusion. In the present study, we examined amoebae that were at different stages of expelling partially exposed nuclei, to follow the sequence of events during the apparent new membrane formation. Unexpectedly, we found that a new membrane is not formed around the nucleus from inside but a hole is sealed primarily by a constriction of the existing membrane, and that cytoplasmic filaments are responsible for the prevention of the loss of cytoplasm.     

Oxygen consumption of human blood platelets. II. Effect of inhibitors on thrombin-induced oxygen burst

The effect of selected inhibitors on the thrombin-stimulated burst and the basal oxygen consumption of washed human platelets were investigated and compared with inhibition of the release reaction. Cyanide (0.2 mM) caused complete inhibition of the basal respiration, but only 15% inhibition of the thrombin-stimulated burst of oxygen consumption. Similar differential inhibitory effects were observed with oligomycin, antimycin, rotenone and N-ethylmaleimide. Prostaglandin E₁ (0.03 mM) and acetylsalicylic acid (0.8 mM) had little effect on basal respiration, but inhibited the thrombin-stimulated burst of oxygen consumption. N-Ethylmaleimide (0.4 mM) inhibited the release of calcium from platelets by 90%, while prostaglandin E₁, acetylsalicylic acid and the above mitochondrial inhibitors caused no more than 30% inhibition of the release reaction. Our results provide evidence that basal respiration and a portion of the thrombin-stimulated burst of oxygen consumption are involved in respiratory chain phosphorylation, and that this component of the thrombin-stimulated burst may be coupled to the maintenance of the release reaction.