Further evidence that eugenol does not bind to DNA in vivo

The naturally-occurring alkenylbenzene, eugenol, was examined for its ability to form DNA adducts in the livers of mice that had been treated with up to 10 mg of the compound. No adducts were detected by 32P-postlabelling with a limit of detection of 1 adduct in 10(9) nucleotides. Under these conditions adducts were readily detected in liver DNA from the structurally-related hepatocarcinogen safrole.     

Evidence that queens do not influence nestmate recognition in Argentine ants

Summary Introduced populations of many invasive ants exhibit low levels of intraspecific aggression. Argentine ants (Linepithema humile), for example, maintain expansive supercolonies in many parts of their introduced range. Recent studies demonstrate that variation in nestmate recognition in L. humile can derive from both environmental and genetic sources. In some ants, pheromones emitted by queens also influence nestmate-recognition behavior. To test if such a phenomenon occurs in Argentine ants, we examined whether levels of intraspecific aggression vary as a function of queen presence or absence in experimental lab colonies. For each of four known supercolonies from southwestern California, we set up a pair of experimental colonies and randomly assigned replicates within each pair to treatment (queen removal) and control (no queen removal) groups. Using two different behavioral assays, we then measured aggressive behavior for ten days, removed queens from colonies in the treatment group, and continued to monitor aggression in both experimental groups for an additional 65 days. Both assays yielded qualitatively similar results: intraspecific aggression remained high throughout the experiment in both experimental groups. These results suggest that L. humile queens fail to influence levels of intraspecific aggression in introduced populations.Received 2 June 2003; revised 1 September 2003; accepted 18 September 2003.     

Synthesis and evaluation of peptidomimetics that bind DNA

A peptidomimetic template, consisting of a hydrophobic scaffold, a dansyl fluorophore, and an Arg-His recognition strand, was tested as a simple mimic of zinc finger 2 of the Zif268 protein. Association constants (K(A)'s) were on the order of 10(5) M(-1) for complexes formed between the mimetic and duplexes d(CGGGAATTCCCG)(2) and d(AAAAAAAAATTTTTTTTT)(2). Modest selectivity was observed for the GC-rich DNA in a 0.5M NaCl/buffer (0.1M phosphate, pH 7.0) solution. Differences in K(A)'s along with observed CD profiles suggest that the mimetic associated with the duplexes using different binding modes. The DNA duplexes had weaker interactions with the free Arg-His recognition strand, the dansyl functional group, and a scaffold that contained only glycines as the recognition strand. The scaffold most likely provides for greater van der Waal's interactions, a larger hydrophobic effect upon association, and reduces the freedom of motion of the side chains. This last effect was confirmed by molecular mechanics calculations and by the fact that the mimetic suffered a smaller loss of entropic energy upon association than the free recognition strand. These studies show that the synthetic scaffold is a promising platform in which peptides can be attached to increase their affinity and possibly selectivity for DNA targets.     

DNA strand specificity in promoter recognition by RNA polymerase.

DNA strand and enzyme subunit specificities involved in the interaction between E. coli RNA polymerase and T7 DNA were studied by photo-crosslinking techniques. In non-specific enzyme-DNA complexes, subunits, sigma, beta, and beta' were crosslinked to both strands of the DNA. Under conditions leading to specific enzyme-promoter complexes, however, only sigma and beta subunits were crosslinked. The sigma subunit was crosslinked preferentially to the non-sense strand at promoter sites. No such strand specificity was observed for the beta subunit. These results provide insight into the molecular mechanism of promoter recognition and indicate that the interaction between RNA polymerase and DNA template is different at promoters and at non-specific sites.     

Mammalian SMC3 C-terminal and coiled-coil protein domains specifically bind palindromic DNA, do not block DNA ends, and prevent DNA bending

The C-terminal domains of yeast structural maintenance of chromosomes (SMC) proteins were previously shown to bind double-stranded DNA, which generated the idea of the antiparallel SMC heterodimer, such as the SMC1/3 dimer, bridging two DNA molecules. Analysis of bovine SMC1 and SMC3 protein domains now reveals that not only the C-terminal domains, but also the coiled-coil region, binds DNA, while the N terminus is inactive. Duplex DNA and DNA molecules with secondary structures are highly preferred substrates for both the C-terminal and coiled-coil domains. Contrasting other cruciform DNA-binding proteins like HMG1, the SMC3 C-terminal and coiled-coil domains do not bend DNA, but rather prevent bending in ring closure assays. Phosphatase, exonuclease, and ligase assays showed that neither domain renders DNA ends inaccessible for other enzymes. These observations allow modifications of models for SMC-DNA interactions.     

The slug parasitic nematode Phasmarhabditis hermaphrodita associates with complex and variable bacterial assemblages that do not affect its virulence

Phasmarhabditis hermaphrodita is a nematode parasite of slugs that is commercially reared in monoxenic culture with the bacterium Moraxella osloensis and sold as a biological molluscicide. However, its bacterial associations when reared in vivo in slugs are unknown. We show that when reared in vivo in slugs, P. hermaphrodita does not retain M. osloensis and associates with complex and variable bacterial assemblages that do not influence its virulence. This is in marked contrast to the entomopathogenic nematodes that form highly specific mutualistic associations with Enterobacteriaceae that are specifically retained during in vivo growth.     

Single-stranded DNA aptamers that bind differentiated but not parental cells: subtractive systematic evolution of ligands by exponential enrichment

In this paper, single-stranded (ss)DNA aptamers with capability to distinguish differentiated PC12 cells from normal PC12 cells were selected by subtractive systematic evolution of ligands by exponential enrichment (SELEX) method. Before each round of selection, randomized ssDNAs were incubated with regular PC12 cells to eliminate those that recognize the common cellular components of both differentiated and undifferentiated PC12 cells. After six rounds of cell-based selection, both of individual aptamers and aptamers of the sixth round pool were found binding to differentiated PC12 cells, but not to the parental PC12 cells. The aptamers of the starting pool showed no such binding. Sequence analysis illustrated that the amount of G content in central random region of these aptamers was much higher than that of the starting pool, which would be expected to be average. The aptamers obtained from this method were also able to identify differentiated PC12 cells from a mixture of both normal and differentiated cells. The results indicate that subtractive SELEX is a useful tool in finding ligands to specific biological markers that distinguish a subtype of cells from cells of homologous origin, such as carcinoma cells among normal epithelial tissues. Both these aptamers and their markers may play important roles in basic research and clinical diagnosis.     

DNA lesion recognition by the bacterial repair enzyme MutM

MutM is a bacterial DNA glycosylase that removes the mutagenic lesion 8-oxoguanine (oxoG) from duplex DNA. The means of oxoG recognition by MutM (also known as Fpg) is of fundamental interest, in light of the vast excess of normal guanine bases present in genomic DNA. The crystal structure of a recognition-competent but catalytically inactive version of MutM in complex with oxoG-containing DNA reveals the structural basis for recognition. MutM binds the oxoG nucleoside in the syn glycosidic configuration and distinguishes oxoG from guanine by reading out the protonation state of the N7 atom. The segment of MutM principally responsible for oxoG recognition is a flexible loop, suggesting that conformational mobility influences lesion recognition and catalysis. Furthermore, the structure of MutM in complex with DNA containing an alternative substrate, dihydrouracil, demonstrates how MutM is able to recognize lesions other than oxoG.     

Binding of mitochondrial leader sequences to Tom20 assessed using a bacterial two-hybrid system shows that hydrophobic interactions are essential and that some mutated leaders that do not bind Tom20 can still be imported

Previous studies pointed to the importance of leucine residues in the binding of mitochondrial leader sequences to Tom20, an outer membrane protein translocator that initially binds the leader during import. A bacteria two-hybrid assay was here employed to determine if this could be an alternative way to investigate the binding of leader to the receptor. Leucine to alanine and arginine to glutamine mutations were made in the leader sequence from rat liver aldehyde dehydrogenase (pALDH). The leucine residues in the C-terminal of pALDH leader were found to be essential for TOM20 binding. The hydrophobic residues of another mitochondrial leader F1beta-ATPase that were important for Tom20 binding were found at the C-terminus of the leader. In contrast, it was the leucines in the N-terminus of the leader of ornithine transcarbamylase that were essential for binding. Modeling the peptides to the structure of Tom20 showed that the hydrophobic residues from the three proteins could all fit into the hydrophobic binding pocket. The mutants of pALDH that did not bind to Tom20 were still imported in vivo in transformed HeLa cells or in vitro into isolated mitochondria. In contrast, the mutant from pOTC was imported less well ( approximately 50%) while the mutant from F1beta-ATPase was not imported to any measurable extent. Binding to Tom20 might not be a prerequisite for import; however, it also is possible that import can occur even if binding to a receptor component is poor, so long as the leader binds tightly to another component of the translocator.