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In mammals, cyclic GMP–AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide 2′3′-cGAMP in response to cytosolic DNA and this triggers an antiviral immune response. cGAS belongs to a large family of cGAS/DncV-like nucleotidyltransferases that is present in both prokaryotes 1 and eukaryotes 2 – 5 . In bacteria, these enzymes synthesize a range of cyclic oligonucleotides and have recently emerged as important regulators of phage infections 6 – 8 . Here we identify two cGAS-like receptors (cGLRs) in the insect Drosophila melanogaster . We show that cGLR1 and cGLR2 activate Sting- and NF-κB-dependent antiviral immunity in response to infection with RNA or DNA viruses. cGLR1 is activated by double-stranded RNA to produce the cyclic dinucleotide 3′2′-cGAMP, whereas cGLR2 produces a combination of 2′3′-cGAMP and 3′2′-cGAMP in response to an as-yet-unidentified stimulus. Our data establish cGAS as the founding member of a family of receptors that sense different types of nucleic acids and trigger immunity through the production of cyclic dinucleotides beyond 2′3′-cGAMP. Two cGAS-like receptors, cGLR1 and cGLR2, identified in Drosophila melanogaster are shown to induce antiviral immunity in response to RNA or DNA virus infections through the production of 2′3′-cGAMP and 3′2′-cGAMP.

A two-marker combination of plastid rbcL and matK has previously been recommended as the core plant barcode, to be supplemented with additional markers such as plastid trnH–psbA and nuclear ribosomal internal transcribed spacer (ITS). To assess the effectiveness and universality of these barcode markers in seed plants, we sampled 6,286 individuals representing 1,757 species in 141 genera of 75 families (42 orders) by using four different methods of data analysis. These analyses indicate that (i) the three plastid markers showed high levels of universality (87.1–92.7%), whereas ITS performed relatively well (79%) in angiosperms but not so well in gymnosperms; (ii) in taxonomic groups for which direct sequencing of the marker is possible, ITS showed the highest discriminatory power of the four markers, and a combination of ITS and any plastid DNA marker was able to discriminate 69.9–79.1% of species, compared with only 49.7% with rbcL + matK; and (iii) where multiple individuals of a single species were tested, ascriptions based on ITS and plastid DNA barcodes were incongruent in some samples for 45.2% of the sampled genera (for genera with more than one species sampled). This finding highlights the importance of both sampling multiple individuals and using markers with different modes of inheritance. In cases where it is difficult to amplify and directly sequence ITS in its entirety, just using ITS2 is a useful backup because it is easier to amplify and sequence this subset of the marker. We therefore propose that ITS/ITS2 should be incorporated into the core barcode for seed plants.

GH3 amino acid conjugases have been identified in many plant and bacterial species. The evolution of GH3 genes in plant species is explored using the sequenced rosids Arabidopsis , papaya, poplar, and grape. Analysis of the sequenced non-rosid eudicots monkey flower and columbine, the monocots maize and rice, as well as spikemoss and moss is included to provide further insight into the origin of GH3 clades. Comparison of co-linear genes in regions surrounding GH3 genes between species helps reconstruct the evolutionary history of the family. Combining analysis of synteny with phylogenetics, gene expression and functional data redefines the Group III GH3 genes, of which AtGH3.12/PBS3, a regulator of stress-induced salicylic acid metabolism and plant defense, is a member. Contrary to previous reports that restrict PBS3 to Arabidopsis and its close relatives, PBS3 syntelogs are identified in poplar, grape, columbine, maize and rice suggesting descent from a common ancestral chromosome dating to before the eudicot/monocot split. In addition, the clade containing PBS3 has undergone a unique expansion in Arabidopsis , with expression patterns for these genes consistent with specialized and evolving stress-responsive functions.

Ubiquitin-related modifier 1 (Urm1) is a ubiquitin-like molecule (UBL) with the dual capacity to act both as a sulphur carrier and posttranslational protein modifier. Here we characterize the Drosophila melanogaster homologues of Urm1 ( CG33276 ) and its E1 activating enzyme Uba4 ( CG13090 ), and show that they function together to induce protein urmylation in vivo. Urm1 conjugation to target proteins in general, and to the evolutionary conserved substrate Peroxiredoxin 5 (Prx5) specifically, is dependent on Uba4. A complete loss of Urm1 is lethal in flies, although a small number of adult zygotic Urm1 n123 mutant escapers can be recovered. These escapers display a decreased general fitness and shortened lifespan, but in contrast to their S. cerevisiae counterparts, they are resistant to oxidative stress. Providing a molecular explanation, we demonstrate that cytoprotective JNK signaling is increased in Urm1 deficient animals. In agreement, molecular and genetic evidence suggest that elevated activity of the JNK downstream target genes Jafrac1 and gstD1 strongly contributes to the tolerance against oxidative stress displayed by Urm1 n123 null mutants. In conclusion, Urm1 is a UBL that is involved in the regulation of JNK signaling and the response against oxidative stress in the fruit fly.

Homologous recombination events between circular chromosomes, occurring during or after replication, can generate dimers that need to be converted to monomers prior to their segregation at cell division. In Escherichia coli, chromosome dimers are converted to monomers by two paralogous site-specific tyrosine recombinases of the Xer family (XerC/D). The Xer recombinases act at a specific dif site located in the replication termination region, assisted by the cell division protein FtsK. This chromosome resolution system has been predicted in most Bacteria and further characterized for some species. Archaea have circular chromosomes and an active homologous recombination system and should therefore resolve chromosome dimers. Most archaea harbour a single homologue of bacterial XerC/D proteins (XerA), but not of FtsK. Therefore, the role of XerA in chromosome resolution was unclear. Here, we have identified dif-like sites in archaeal genomes by using a combination of modeling and comparative genomics approaches. These sites are systematically located in replication termination regions. We validated our in silico prediction by showing that the XerA protein of Pyrococcus abyssi specifically recombines plasmids containing the predicted dif site in vitro. In contrast to the bacterial system, XerA can recombine dif sites in the absence of protein partners. Whereas Archaea and Bacteria use a completely different set of proteins for chromosome replication, our data strongly suggest that XerA is most likely used for chromosome resolution in Archaea.

Plant ADP-glucose pyrophosphorylase (AGP) is a heterotetrameric enzyme composed of two large and two small subunits. Here, we report the structures of the maize (Zea mays) genes encoding AGP small subunits of leaf and endosperm. Excluding exon 1, protein-encoding sequences of the two genes are nearly identical. Exon 1 coding sequences, however, possess no similarity. Introns are placed in identical positions and exhibit obvious sequence similarity. Size differences are primarily due to insertions and duplications, hallmarks of transposable element visitation. Comparison of the maize genes with other plant AGP small subunit genes leads to a number of noteworthy inferences concerning the evolution of these genes. The small subunit gene can be divided into two modules. One module, encompassing all coding information except that derived from exon 1, displays striking similarity among all genes. It is surprising that members from eudicots form one group, whereas those from cereals form a second group. This implies that the duplications giving rise to family members occurred at least twice and after the separation of eudicots and monocot cereals. One intron within this module may have had a transposon origin. A different evolutionary history is suggested for exon 1. These sequences define three distinct groups, two of which come from cereal seeds. This distinction likely has functional significance because cereal endosperm AGPs are cytosolic, whereas all other forms appear to be plastid localized. Finally, whereas barley (Hordeum vulgare) reportedly employs only one gene to encode the small subunit of the seed and leaf, maize utilizes the two genes described here.

dTDP-L-Rhamnose biosynthetic gene cluster was cloned from Thermus caldophilus. A cluster of four open reading frames, strmlA, B, C and D, responsible for the production of dTDP-L-rhmanose, was screened from the genomic library. Thermophilic glucose-1-phosphate thymidylyltransferase, encoding 356 amino acids with a calculated molecular weight 38 kDa, was expressed under the control of the tac promoter in E. coli. The expressed enzyme, stRmlA is thermostable up to 70 degrees C and apparently retained its activity even up to 90 degrees C. It shares 73% sequence identity to glucose-1-phosphate thymidylyltransferase from Streptomyces argillaceus. Amino acid sequence comparison of stRmlA with ten glucose-1-phosphate thymidylyltransferases indicated higher number of unusual hydrophobic residues (A10, A58, A69, A252, V225, V257, V265, 1242 and 1246) and charged residues (D43, D160, D248, D249, D315, H124, H201, H283 and H354) in stRmlA.

The Holliday junction, a key intermediate in both homologous and site-specific recombination, is generated by the reciprocal exchange of single strands between two DNA duplexes. Resolution of the junctions can occur in two directions with respect to flanking markers, either restoring the parental DNA configuration or generating a genetic crossover. Recombination can be regulated, in principle, by factors that influence the directionality of the resolution step. We demonstrate that the vaccinia virus DNA topoisomerase, a eukaryotic type I enzyme, catalyzes resolution of synthetic Holliday junctions in vitro. The mechanism entails concerted transesterifications at two recognition sites, 5′-CCCTT $\\downarrow $, that are opposed within a partially mobile four-way junction. Cruciforms are resolved unidirectionally and with high efficiency into two linear duplexes. These findings suggest a model whereby type I topoisomerases may either promote or suppress genetic recombination in vivo.

Bacterial family C DNA polymerases (DNA pol IIIs), the major chromosomal replicative enzymes, have been provisionally classified based on primary sequences and domain structures into three classes: class I (Escherichia coli DNA pol C-type), class II (Bacillus subtilis DNA pol C-type), and class III (cyanobacterial DNA pol C-type), respectively. We have sequenced the structural gene encoding the DNA pol C catalytic subunit of the thermophilic bacterium Thermus aquaticus. This gene, designated the Taq DNA pol C gene, contains a 3660-bp open reading frame which specifies a polypeptide of molecular weight of 137,388 daltons. Comparative sequence analyses revealed that Taq DNA pol C is a class I family C DNA polymerase. The Taq DNA pol C is most closely related to the Deinococcus radiodurans DNA pol C. Although a phylogenetic tree based on the class I family C DNA pols is still in the provisional stage, some important conclusion can be drawn. First, the high-G+C and the low-G+C Gram-positive bacteria are not monophyletic. Second, the low-G+C Gram-positive bacteria contain multigenes of family C DNA pols (classes I and II). Third, the cyanobacterial family C DNA pol, classified as class III because it is encoded by a split gene, forms a group with the high-G+C Gram-positive bacteria.

The diversity of resolvase (tnpR) genes carried by a number of mercury resistant soil bacteria has been investigated by DNA sequencing. The resulting DNA sequence information was compared to previously published tnpR DNA sequences and to previously published restriction fragment length polymorphism (RFLP) data, permitting the relationships between DNA sequencing and RFLP approaches to be studied by the use of phylogenetic trees. DNA maximum likelihood and DNA parsimony were used to construct a variety of phylogenetic trees. DNA sequencing confirmed the validity of RFLP analysis and highlighted the importance of restriction endonuclease choice upon the resulting RFLP patterns and dendrogram topology. The tnpR genes of two previously uncharacterised mercury resistant bacteria, T2-7 and T2-12 were also studied. DNA sequence data placed T2-7 in a previously described gene class, tnpR-D and T2-12 in a new gene class, tnpR-F. The significance of this data with respect to the recombination and evolution events occurring within bacterial populations are discussed.