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.     

ASYMMETRICAL DEVELOPMENTAL PLASTICITY IN AN INTERTIDAL SNAIL

Field and laboratory experiments were used to investigate the variation and phenotypic plasticity in the adhesive abilities of the intertidal snail Nucella lapillus between high- and low-wave-energy environments. Whelks from an exposed coast produced a larger pedal surface area and were more resistant to dislodgement than were similar-sized individuals from a protected shore. Tenacity (g/cm²) was similar between individuals from exposed and protected shores, indicating that variation in resisting dislodgement was solely a function of pedal surface area. Whelks from exposed and protected shores did not differ in pedal surface area as they emerged from egg capsules or when reared in the laboratory under uniform conditions, suggesting that variation between populations does not represent genetic differentiation. Individuals from high-and low-wave-energy environments reared intertidally produced a larger pedal surface area than did those reared in the laboratory. The extent to which pedal surface area increased corresponded to the intensity of wave action. These findings suggest that pedal surface area is a highly plastic character modulated or induced by the water turbulence accompanying breaking waves. A reciprocal-transplant experiment confirmed this notion but revealed an asymmetry in the plasticity. Snails from the protected site transplanted to an exposed shore formed a much larger pedal surface area than did controls reared on the protected shore. In contrast, whelks from a wave-swept shore transplanted to a protected shore differed little from their controls reared on the exposed shore. The asymmetric response parallels a possible asymmetry in the risks of acclimating to a temporally unpredictable environmental cue, such as wave action.     

Transgenic HLA-DR alpha faithfully reconstitutes IE-controlled immune functions and induces cross-tolerance to E alpha in E alpha 0 mutant mice

We have constructed transgenic mice that express the human class II MHC molecule HLA-DR alpha on a genetic background in which the equivalent endogenous gene, H-2 IE alpha, is not expressed. In these mice, DR alpha complemented the E beta chain such that tissue-specific expression of an interspecies hybrid DR alpha-E beta heterodimer was obtained. Despite 25% amino acid differences between DR alpha and E alpha, immune responsiveness to IE-controlled antigens, clonal deletion of IE-reactive T cells, and alloantigenicity were quantitatively and qualitatively indistinguishable in IE-positive mice and in mice that had integrated at least four copies of the transgene. These results demonstrate a remarkable degree of structural, regulatory, and functional conservation. They also suggest that tolerance induction involves only discrete portions of MHC molecules.     

Interaction of (Na + K + 2 Cl) cotransport and the Na/K pump in cultured chick cardiac myocytes

We have recently reported the presence of an electroneutral (Na + K + 2 Cl) cotransport mechanism that is bumetanide-sensitive and maintains Cl_i above its electrochemical equilibrium in cultured chick heart cells. In steady state, (Na + K + 2 Cl) cotransport is inwardly directed and so contributes to the Na influx that must be counterbalanced by the activity of the Na/K pump to maintain Na_i homeostasis. We now show that manipulating (Na + K + 2 Cl) cotransport by restoring Cl_o to a Cl-free solution indirectly influences Na/K pump activity because the bumetanide-sensitive recovery of a _infNa ^supi to its control level and the accompanying hyperpolarization could be blocked by 10^–4M ouabain. In another protocol, when the Na/K pump was reactivated by restoring K_o (from 0.5 mM to 5.4 mM) and removing ouabain, the recovery of a_Na was attenuated by 10^–4M bumetanide. The relatively slow rate of ouabain dissociation coupled with the activation of Na influx by (Na + K + 2 Cl) cotransport clearly establishes the interaction of these transport mechanisms in regulating Na_i. Although (Na + K + 2 Cl) cotransport is electroneutral, secondary consequences of its activity can indirectly affect the electrophysiological properties of cardiac cells.     

The fine structure of the newly discovered propodial ganglia of the veliger larva of the nudibranch Onchidoris bilamellata

Summary Two pairs of ganglia are found in the propodial region of the veliger of Onchidoris bilamellata: the anterolateral pair is located at the foremost corners of the propodium, and the frontal pair is located beside the propodial midline. Both sets of ganglia are positioned below the epidermis, and they are joined to the cerebral ganglia by large, common connectives. Each ganglion possesses sensory cells, nerve cells and sheath cells, and the frontal pair contains a complement of secretory cells. Externally, the propodial ganglia are manifested as sensory fields. The fields of the anterolateral pair are elliptical in shape, and each appears as a band of cilia bordering an unciliated zone. The region devoid of cilia is composed of ordinary epidermal cells, whereas the ciliated portion is comprised of dendritic endings originating from cells in the ganglion. Dendrites arise from one type of sensory cell and pass through the epidermis in bundles. Each dendrite terminates as a single cilium at the epidermal surface. Sensory fields of the frontal ganglia are key-shaped and oppose one another on the anterior end of the foot. Each field appears as a flat, circular, unciliated region which extends into a ciliated groove that runs dorsally toward the mouth. The groove contains the terminals of secretory cells, ciliated sensory cells, and the cell bodies of nonciliated sensory cells. The nonciliated sensory cells, characterized by a microvillous apex, are the dominant cells in the flattened circular zone. The space between the frontal ganglia and the epidermis is bridged by bundles of processes which are similar to those of the anterolateral ganglia. However, these tracts contain collections of the apical processes of secretory cells, the dendrites of ciliated sensory cells, and the axons of nonciliated sensory cells. Morphological and behavioral evidence indicates that the propodial ganglia serve a chemosensory function during settlement and metamorphosis.     

Two Developmentally Regulated Isoenzymes of Calmodulin-Stimulated Protein Kinase II in Rat Forebrain

Soluble calmodulin-stimulated protein kinase II has been purified from adult and 10-day-old rat forebrain. By autoradiography, the alpha/beta subunit ratios of the 10-day and adult enzymes were 0.67 +/- 0.03 and 2.20 +/- 0.15, respectively. By silver staining, the alpha/beta subunit ratios were 1.02 +/- 0.06 and 2.36 +/- 0.10, respectively. The apparent holoenzyme molecular masses of the purified 10-day and adult enzymes were 500,000 daltons and 700,000 daltons. However, varying the purification conditions revealed higher and lower molecular mass forms at both ages and suggested that the form of the kinase that is usually purified is merely that which has the highest affinity for calmodulin-Sepharose and may not be the form of the kinase that exists in vivo. The subunits of the adult and 10-day enzymes were indistinguishable by one- and two-dimensional electrophoresis and one-dimensional proteolytic peptide maps. These results are consistent with the suggestion that at least two developmentally regulated isoenzymes of this kinase exist in rat forebrain.     

Anti-Ig-mediated proliferation of human B cells in the absence of protein kinase C

Cross-linking of surface Ig has been shown to stimulate phosphatidylinositol hydrolysis in murine B cells, leading to increases in [Ca2+]i and activation of protein kinase C (PKC). Preliminary evidence suggests that a similar activation mechanism occurs in human B cells. We wished to examine whether anti-Ig antibody-stimulated human B cell proliferation is as dependent upon the presence of PKC as is anti-Ig-mediated murine B cell proliferation. Using highly purified, small, dense peripheral-blood B lymphocytes from healthy adult donors, we confirmed that PMA, a direct activator of PKC, is a potent mitogen for human B cells that synergizes with anti-mu antibody. Furthermore, we demonstrated that PMA treatment abolishes detectable cellular stores of immunoreactive PKC. However, after such depletion of cellular PKC, anti-mu antibody is still capable of delivering a proliferative signal to human B cells. It is unlikely that this signal occurs solely on the basis of increases in [Ca2+]i, because the calcium ionophore A23187 does not induce a proliferative response in PMA-treated B cells similar in magnitude to that seen with anti-mu. Additionally, the finding that pretreatment of B cells with PMA ablates the ability of anti-Ig antibody to mobilize intracellular and extracellular calcium also suggests that the ability of PMA to enhance anti-Ig mediated stimulation does not depend on elevations of [Ca2+]i induced by anti-Ig. Together, these observations suggest that anti-Ig signaling of human B cells may occur via other pathways in addition to the phosphatidylinositol system of calcium influx and PKC activation.     

Beta-adrenergic receptor characteristics of postnatal rat myocardial cell preparations

Summary Primary mycolardial cell cultures and freshly isolated cardiac cells in suspension resprensent two isolated, whole cell models for investigating cellular transsarcolemmal⁴⁵Ca⁺⁺ exchange in response to a receptor-coupled stimulus. Studies were performed to characterize beta-adrenergic receptor binding, beta-adrenergic receptor mediated cellular calcium (⁴⁵Ca⁺⁺) exchange, and viability in purified primary myocardial cell cultures and freshly isolated cardiac cells in suspension obtained from 3-to 3-d-old Sprague-Dawley rats. In addition, beta-adrenergic receptor binding was characterized in whole-heart crude membrane preparations. All three preparations had saturable beta-adrenergic binding sites with the antagonist [¹²⁵I]iodopindolol ([¹²⁵I]IPIN). The suspensions had a significantly lower B _max (42±6 fmol/mg protein) than the membranes and cultures (77±8 and 95±10 fmol/mg protein, respectively). The K _D of the cultures (218±2.0 pM) was significantly higher than that for the suspensions (107 ±1.3 pM) and membranes (93±1.3 pM). Viability was significantly lower in the suspensions (57%) when compared to 94% viability in myocardial cell cultures after 3 h of incubation in Kreb's Henseleit buffer. Incubation of the cultures with 5.0×10⁻⁷ M isoproterenol resulted in a significant increase in⁴⁵Ca⁺⁺ exchange as early as 15 s. In contrast,⁴⁵Ca⁺⁺ exchange into the suspensions was not increased. Although both primary cell cultures and cardiac cells in suspension possess saturable beta-adrenergic receptors, only the monolayer cultures exhibited functional beta-adrenergic receptor-mediated⁴⁵Ca⁺⁺ exchange. Of the two intact cell models investigated, these data suggest that primary myocardial cell cultures are more suitable than cell suspensions for investigating beta-adrenergic receptor binding and functions in the postnatal rat heart. This research was supported by The University of Texas Research Institute, a grant from the Texas Advanced Research Technology Program awarded to S. W. Leslie and R. E. Wilcox, and contract 223-86-2109 from the Food and Drug Administration.