A Requirement for Argonaute 4 in Mammalian Antiviral Defense

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Large non-homology in heteroduplex DNA is processed differently than single base pair mismatches

Summary Unmethylated DNA heteroduplexes with a large single stranded loop in one strand have been prepared from separated strands of DNA from two different strains of bacteriophage , one of which has a 800 base pair IS1 insertion in the cI gene. The results of transfections with these heteroduplexes into wild-type and mismatch repair deficient bacteria indicate that such large non-homologies are not repaired by the Escherichia coli mismatch repair system. However, the results do suggest that some process can act to repair such large non-homologies in heteroduplex DNA. Transfections of a series of recombination and excision repair deficient mutants suggest that known excision or recombination repair systems of E. coli are not responsible for the repair. Repair of large non-homologies may play a role in gene conversion involving large insertion or deletion mutations.     

Isolation and characterisation of transposon-induced mutants of Erwinia carotovora subsp. atroseptica exhibiting reduced virulence

Summary The blackleg pathogen Erwinia carotovora subsp. atroseptica (Eca) causes an economically important disease of potatoes. We selected a genetically amenable Eca strain for the genetic analysis of virulence. Tn5 mutagenesis was used to generate nine mutants which exhibited reduced virulence (Rvi^-) of strain SCRI1043. Following physiological characterisation, mutants were divided into three classes: (1) auxotrophs; (2) extracellular enzyme mutants; and (3) a growth rate mutant. The isolation of these Rvi^- mutants has allowed us to consider some factors that affect Eca virulence.     

Cloning and DNA sequence of plasmid determinant iss, coding for increased serum survival and surface exclusion, which has homology with lambda DNA

Summary Escherichia coli K12 cells carrying a cloned 1.4 kb HindIII fragment from plasmid ColV2-K94, showed increased survival in guinea pig serum. The recombinant plasmid also conferred group II surface exclusion, i.e. the cells were reduced in recipient ability towards the incoming plasmid R538drd in conjugation experiments. Southern blotting suggested homology with bacteriophage lambda DNA and to the insertion element IS2. Determination of the DNA sequence of the fragment demonstrated the presence of a truncated IS2 (165 bp), separated by 250 bp from a 900 bp stretch of homology with lambda DNA, beginning within the Rz gene and continuing in the rightward direction on the lambda map. A 97 amino acid open reading frame (ORF) adjacent to Rz and on the opposite strand, remained intact in iss, with several amino acid changes. The ORF in iss is preceded by sequences resembling prokaryotic ribosome binding sites and promoters.     

Cell-membrane phospholipase C is involved in inducing the antiviral effect of interferon

A monospecific inhibitory antibody directed to phospholipase C (phosphoinositidase C) blocked the antiviral effect of human interferons alpha and beta when tested on human quiescent fibroblasts challenged with the vesicular stomatitis virus. This action was due to specific inhibition of polyphosphoinositide hydrolysis because (a) the F(ab)₂ fragment of the antibody molecule was also inhibitory; (b) excess antibodies directed to phospholipase A₂ and to a phosphatidylcholine-preferring phospholipase C did not have any inhibitory effect, and (c) the combination of 12-O-tetradecanoylphorbol-acetate and calcium ionophore A23187 had an interferon-like antiviral effect which was not influenced by the inhibitory anti-phospholipase C antibodies. To avoid an interferon-like effect due to induction of interferon by second messengers, Vero cells, which lack interferon biosynthesis, were also used. Liposomes containing inositol 1,4,5-triphosphate and 1-oleoyl-2-acetyl-rac-glycerol protected Vero cells against the infection with the vesicular stomatitis virus. These results taken together show that phosphoinositide-derived second messengers are involved in triggering the antiviral effect of interferons alpha and beta.     

Mechanisms of degradation of 2′-5′ oligoadenylates

We have studied the mechanisms of breakdown of 2'-5' oligoadenylates. We monitored the time-courses of degradation of ppp(A2'p5')nA (dimer to tetramer) and of 5'OH-(A2'p5')nA (dimer to pentamer) in unfractionated L1210 cell extract. The 5' triphosphorylated 2'-5' oligoadenylates are converted by a phosphatase activity. However, 2'-5' oligoadenylates are degraded mainly by phosphodiesterase activity which splits the 2'-5' phosphodiester bond sequentially at the 2' end to yield 5' AMP and one-unit-shorter oligomers. The nonlinear least-squares curve-fitting program CONSAM was used to fit these kinetics and to determine the degradation rate constant of each oligomer. Trimers and tetramers, whether 5' triphosphorylated or not, are degraded at the same rate, whereas 5' triphosphorylated dimer is rapidly hydrolyzed and 5'-OH dimer is the most stable oligomer. The interaction between degradation enzymes and the substrate strongly depends on the presence of a 5' phosphate group in the vicinity of the phosphodiester bond to be hydrolyzed; indeed, when this 5' phosphate group is present, as in pp/pA2'p5'A/or A2'/p5'A2'p5'A/, affinity is high and maximal velocity is low. Such a degradation pattern can control the concentration of 2'-5' oligoadenylates active on RNAse L either by limiting their synthesis (5' triphosphorylated dimer is the primer necessary for the formation of longer oligomers) and/or by converting them into inhibitory (e.g., monophosphorylated trimer) or inactive (e.g., nonphosphorylated oligomers) molecules.     

Bacteriophage lambda DNA packaging: A mutant terminase that is independent of integration host factor

Summary ⁺ is able to grow in Escherichia coli cells lacking integration host factor (IHF), producing a burst of approximately 25% that produced in IHF⁺ cells. In vitro, however, we find that the DNA packaging enzyme terminase is strongly dependent on IHF in both cos cleavage reactions and DNA packaging reactions. The cos59 mutation renders dependent on IHF in vivo. The cos59 mutation is a deletion of 3 base pairs at the XmnI site in the cohesive end site (cos) of . Variants of cos59 that were able to grow in the absence of IHF were isolated and found to carry a mutation, called ms1, in the Nu1 gene, which codes for the small subunit of terminase. The Nu1ms1 mutation results in a change of the 40th amino acid of the Nu1 gene product from leucine to phenylalanine. The Nu1ms1 terminase was independent of IHF in packaging reactions in vitro. The results indicate that the mutation either renders terminase: (1) able to utilize some host protein other than IHF, or (2) totally independent of host factors.     

2-Aminopurine and 5-bromouracil induce alleviation of type I restriction in Escherichia coli: Mismatches function as inducing signals?

Summary The EcoK restriction of unmodified phage is 1000-fold alleviated in Escherichia coli grown in the presence of base analogs 2-aminopurine (2AP) and 5-bromouracil (5BU). 2AP treatment of bacteria affects specificially the type I restriction systems (EcoA, EcoB, EcoD and EcoK) and does not influence type II (EcoRI) and type III (EcoP1) restriction. 2AP-induced alleviation of restriction occurs in bacteria which are deficient in the SOS response (recA and lexA) and mismatch repair (mutH, mutL and mutS) and can be distinguished from the alleviation of restriction observed in dam ^- strains. We suggest that mismatches induced by 2AP and 5BU may function as an inducing signal for the alleviation of restriction observed in the presence of base analogs.