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.     

Translational control during the acute phase response. Ferritin synthesis in response to interleukin-1.

Interleukin-1 (IL-1 beta) increases the synthesis of both heavy and light (L)-ferritin subunits when added to human hepatoma cells (HepG2) grown in culture. RNase protection and Northern blot analysis with L-ferritin probes revealed that no changes in L-ferritin mRNA levels occur after cytokine stimulation. However, the induction coincides with an increased association of the L-subunit mRNA with polyribosomes. Since the recruitment of stored ferritin mRNA onto polyribosomes is seen when iron enters the cell, the effect of IL-1 beta on iron uptake was tested and was found to be unaffected by the lymphokine. Neither transferrin receptor mRNA levels nor the number of receptors displayed on the cell surface was affected by IL-1 beta. However, the action of the cytokine on ferritin translation is inhibited by the action of the intracellular iron chelator deferoxamine. These data indicate that IL-1 beta induces ferritin gene expression by translational control of its mRNA. The pathway of induction is different from iron-dependent ferritin gene expression whereas regulation requires the background presence of cellular iron.     

IL-1 induces high affinity IL-2 receptor expression of CD4-8- thymocytes

We investigated the role of cytokines for the growth of CD4-8-thymocytes (double negative thymocytes) (DNT) in vitro and found that IL-1-induced IL-2-dependent proliferation of only the IL-2R-positive DNT subpopulation. The presence of IL-1 during the first 18 h of culture was sufficient for an optimal response and suggested that IL-1 induced DNT differentiation. We could indeed show by RNA dot blot analysis that IL-1 stimulated de novo expression of the p55 chain of the IL-2R thus initiating high affinity IL-2 binding and a proliferative response. Because macrophages and epithelial cells in the thymus produce IL-1 we propose that IL-1 is involved in early events during maturation of immature thymocytes.     

Energy coupling sites in the electron transport chain of Zymomonas mobilis

Abstract Energy-coupling sites in the electron transport chain of the obligately fermentative aerotolerant bacterium Zymomonas mobilis were examined. The H⁺ /O stoichiometry of the electron transport chain in intact bacteria oxidizing ethanol was close to 3.3. Cytoplasmic membrane vesicles coupled NADH oxidation to ATP synthesis. With ascorbate/phenazine methosulfate they showed oxygen uptake which was sensitive to antimycin A, but no significant ATP synthesis could be detected. Cells with a defective coupling site I, prepared by cultivation on a sulfate-deficient medium, showed a decreased rotenone sensitivity of respiration, and they lacked almost all the respiration-driven proton translocation and ATP synthesis. We conclude that, despite the reported composition of the electron transport chain, only energy coupling site 1 was functional in Z. mobilis .     

p103 and A60: novel proteins of the neuronal membrane-associated cytoskeleton.

Associated with the neuronal plasma membrane are cytoskeletal proteins which probably control the specialization of the membrane into axonal and dendritic domains. Specialized isoforms of the proteins spectrin and ankyrin are located in each region and provide molecular mechanisms for locating specific transmembrane proteins at required points. However, spectrin and ankyrin were defined by extensions of the model for the erythrocyte membrane, an analogy unlikely to provide a complete account of the neuronal membrane skeleton. We have defined two new proteins of the neuronal membrane skeleton, designated p103 and A60. p103 is enriched in post-synaptic densities and binds with high affinity to integral membrane proteins--we suggest that it may have a role in linking the cytoskeleton to synaptic glycoproteins. A60 is a 60 kDa axonal protein, which appears to form a lining to the axolemma. It is almost exclusively axonal, although some neurons (such as Purkinje cells) appear to contain it in the cell body and initial dendrite segment. A60 binds both ankyrin and neurofilaments, and may have a role in transmitting information critical to axonal morphology to the membrane.     

Methylation patterns at the hypervariable X-chromosome locus DXS255 (M27 beta): correlation with X-inactivation status.

Methylation patterns surrounding a hypervariable X-chromosome locus, DXS255, have been analyzed with the restriction enzyme MspI and its methylation-sensitive isoschizomer HpaII. HpaII sites flanking the hypervariable region were found to be methylated on 41 active X chromosomes and unmethylated on 11 inactive X chromosomes present in a range of male, female, and hybrid cells and tissues. This differential methylation pattern coupled with the previously described high level (greater than 90%) of heterozygosity at the DXS255 locus can therefore be applied to determine the inactivation status of X chromosomes in females heterozygous for X-linked disease and in tumor clonality studies.     

Lactate from cultures of Lactobacillus casei recovered in a fluidized bed column using ion exchange resin

Summary Lactic acid produced by continuous culture of L.casei in an upflow packed bed reactor, was recovered with Amberlite IRA 400 in a fluidized bed column. Bed expansions of 1.25 and 2.25 were applied. Reutilization did not alter the capability of net recovery of 0.048 ± 0.01 g lactic acid/g resin. When 2200 cm/h of ascensional velocity was used, (bed expansion of 2.25), the resin adsorbed 39.3% of the initial lactic acid and 63.5% was eluted. This resin supported the highest exchange capacity of 0.126 g lactic acid/g resin. Applying high flow rates, the process has potential industrial applications due to the short time employed.