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C1 repressor of phage P1 is inactivated by noncovalent binding of P1 Coi protein.

The temperate phage P1 encodes two genes whose products antagonize the action of the phage's C1 repressor of lytic functions, namely a distantly linked antirepressor gene, ant, and a closely linked c1 inactivator gene, coi. Starting with an inducible coi-recombinant plasmid, Coi protein was overproduced and purified to near homogeneity. By using a DNA mobility shift assay we demonstrate that Coi protein inhibits the operator binding of the C1 repressors of the closely related P1 and P7 phages. Coi protein (Mr = 7,600) exerts its C1-inactivating function by forming a complex with the C1 repressor (Mr = 32,500) at a molar ratio of about 1:1, as shown by density gradient centrifugation and gel filtration. C1 repressor and Coi protein are recovered in active form from the complex, suggesting that noncovalent interactions are the sole requirements for complex formation. The interplay of repressor and antagonists operating in the life cycle of P1 is discussed.     

8-Hydroxypegolettiolide,a sesquiterpene lactone with a new carbon skeleton and further constituents from Pegolettia senegalensis

The investigation of Pegolettia senegalensis afforded several new sesquiterpene lactones, eight cis-6,12-germacra-trans,trans-1(10),4,11-trienolides, five cis-6,12-eudesmanolides, two elemanolides, 8,14-cyclogermacra-1(10),4,7(11)-trien-6,12-olide with a new carbon skeleton, three germacra-1(10),4,11(13)-trien-12-oic acids with ester residues at C-8, 18-hydroxygeranyl nerol, 1,3-dihydroxyoctadecane and a mixture of esters of 3,4-dihydroxy-dihydrocinnamyl alcohol. The structures were elucidated by high field ¹HNMR spectroscopy and some chemical transformations. The C-10 configuration of 6,12-cis-eudesmanolides from Calostephane divaricata and Inula crithmoides most likely has to be corrected. The chemotaxonomic situation of the genus Pegolettia and biogenetic considerations are discussed briefly.     

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Antiphytovirale Aktivität von lipophilen Fraktionen aus der Hefe Lodderomyces elongisporus IMET H 128

Lipid extract isolated from biomasses of Lodderomyces elongisporus IMET H 128 grown on gas oil 7(B.p. 240–380°C) and fractions of the lipid extract (acetone soluble and fatty acid fractions) reduced the concentration of potato virus X in inoculated as well as in secondarily infected leaves markedly. The antiphytoviral inactive phosphatide fraction was converted into a fraction with high activity by partial hydrolysis with SO₂ as acting agent.     

Genome-wide haploinsufficiency screen reveals a novel role for γ-TuSC in spindle organization and genome stability

How subunit dosage contributes to the assembly and function of multimeric complexes is an important question with implications in understanding biochemical, evolutionary, and disease mechanisms. Toward identifying pathways that are susceptible to decreased gene dosage, we performed a genome-wide screen for haploinsufficient (HI) genes that guard against genome instability in Saccharomyces cerevisiae. This led to the identification of all three genes (SPC97, SPC98, and TUB4) encoding the evolutionarily conserved γ-tubulin small complex (γ-TuSC), which nucleates microtubule assembly. We found that hemizygous γ-TuSC mutants exhibit higher rates of chromosome loss and increases in anaphase spindle length and elongation velocities. Fluorescence microscopy, fluorescence recovery after photobleaching, electron tomography, and model convolution simulation of spc98/+ mutants revealed improper regulation of interpolar (iMT) and kinetochore (kMT) microtubules in anaphase. The underlying cause is likely due to reduced levels of Tub4, as overexpression of TUB4 suppressed the spindle and chromosome segregation defects in spc98/+ mutants. We propose that γ-TuSC is crucial for balanced assembly between iMTs and kMTs for spindle organization and accurate chromosome segregation. Taken together, the results show how gene dosage studies provide critical insights into the assembly and function of multisubunit complexes that may not be revealed by using traditional studies with haploid gene deletion or conditional alleles.     

Genomic analysis reveals key aspects of prokaryotic symbiosis in the phototrophic consortium “Chlorochromatium aggregatum”

Background

‘Chlorochromatium aggregatum’ is a phototrophic consortium, a symbiosis that may represent the highest degree of mutual interdependence between two unrelated bacteria not associated with a eukaryotic host. ‘Chlorochromatium aggregatum’ is a motile, barrel-shaped aggregate formed from a single cell of ‘Candidatus Symbiobacter mobilis”, a polarly flagellated, non-pigmented, heterotrophic bacterium, which is surrounded by approximately 15 epibiont cells of Chlorobium chlorochromatii, a non-motile photolithoautotrophic green sulfur bacterium.

Results

We analyzed the complete genome sequences of both organisms to understand the basis for this symbiosis. Chl. chlorochromatii has acquired relatively few symbiosis-specific genes; most acquired genes are predicted to modify the cell wall or function in cell-cell adhesion. In striking contrast, ‘Ca. S. mobilis’ appears to have undergone massive gene loss, is probably no longer capable of independent growth, and thus may only reproduce when consortia divide. A detailed model for the energetic and metabolic bases of the dependency of ‘Ca. S. mobilis’ on Chl. chlorochromatii is described.

Conclusions

Genomic analyses suggest that three types of interactions lead to a highly sophisticated relationship between these two organisms. Firstly, extensive metabolic exchange, involving carbon, nitrogen, and sulfur sources as well as vitamins, occurs from the epibiont to the central bacterium. Secondly, ‘Ca. S. mobilis’ can sense and move towards light and sulfide, resources that only directly benefit the epibiont. Thirdly, electron cycling mechanisms, particularly those mediated by quinones and potentially involving shared protonmotive force, could provide an important basis for energy exchange in this and other symbiotic relationships.     

A Missing PD-L1/PD-1 Coinhibition Regulates Diabetes Induction by Preproinsulin-Specific CD8 T-Cells in an Epitope-Specific Manner

Coinhibitory PD-1/PD-L1 (B7-H1) interactions provide critical signals for the regulation of autoreactive T-cell responses. We established mouse models, expressing the costimulator molecule B7.1 (CD80) on pancreatic beta cells (RIP-B7.1 tg mice) or are deficient in coinhibitory PD-L1 or PD-1 molecules (PD-L1^−/− and PD-1^−/− mice), to study induction of preproinsulin (ppins)-specific CD8 T-cell responses and experimental autoimmune diabetes (EAD) by DNA-based immunization. RIP-B7.1 tg mice allowed us to identify two CD8 T-cell specificities: pCI/ppins DNA exclusively induced K^b/A_12–21-specific CD8 T-cells and EAD, whereas pCI/ppinsΔA_12–21 DNA (encoding ppins without the COOH-terminal A_12–21 epitope) elicited K^b/B_22–29-specific CD8 T-cells and EAD. Specific expression/processing of mutant ppinsΔA_12–21 (but not ppins) in non-beta cells, targeted by intramuscular DNA-injection, thus facilitated induction of K^b/B_22–29-specific CD8 T-cells. The A_12–21 epitope binds K^b molecules with a very low avidity as compared with B_22–29. Interestingly, immunization of coinhibition-deficient PD-L1^−/− or PD-1^−/− mice with pCI/ppins induced K^b/A_12–21-monospecific CD8 T-cells and EAD but injections with pCI/ppinsΔA_12–21 did neither recruit K^b/B_22–29-specific CD8 T-cells into the pancreatic target tissue nor induce EAD. PpinsΔA_12–21/(K^b/B_22–29)-mediated EAD was efficiently restored in RIP-B7.1⁺/PD-L1^−/− mice, differing from PD-L1^−/− mice only in the tg B7.1 expression in beta cells. Alternatively, an ongoing beta cell destruction and tissue inflammation, initiated by ppins/(K^b/A_12–21)-specific CD8 T-cells in pCI/ppins+pCI/ppinsΔA_12–21 co-immunized PD-L1^−/− mice, facilitated the expansion of ppinsΔA_12–21/(K^b/B_22–29)-specific CD8 T-cells. CD8 T-cells specific for the high-affinity K^b/B_22–29- (but not the low-affinity K^b/A_12–21)-epitope thus require stimulatory ´help from beta cells or inflamed islets to expand in PD-L1-deficient mice. The new PD-1/PD-L1 diabetes models may be valuable tools to study under well controlled experimental conditions distinct hierarchies of autoreactive CD8 T-cell responses, which trigger the initial steps of beta cell destruction or emerge during the pathogenic progression of EAD.     

Whole-Genome Sequencing of Aspergillus terreus Species Complex

Mycopathologia - Aspergillus terreus species complex is an opportunistic fungal pathogen increasingly implicated in invasive infection, as well as chronic respiratory disease. Currently, an...