Thermodynamic and kinetic basis for the relaxed DNA sequence specificity of "promiscuous" mutant EcoRI endonucleases

Promiscuous mutant EcoRI endonucleases produce lethal to sublethal effects because they cleave Escherichia coli DNA despite the presence of the EcoRI methylase. Three promiscuous mutant forms, Ala138Thr, Glu192Lys and His114Tyr, have been characterized with respect to their binding affinities and first-order cleavage rate constants towards the three classes of DNA sites: specific, miscognate (EcoRI*) and non-specific. We have made the unanticipated and counterintuitive observations that the mutant restriction endonucleases that exhibit relaxed specificity in vivo nevertheless bind more tightly than the wild-type enzyme to the specific recognition sequence in vitro, and show even greater preference for binding to the cognate GAATTC site over miscognate sites. Binding preference for EcoRI* over non-specific DNA is also improved. The first-order cleavage rate constants of the mutant enzymes are normal for the cognate site GAATTC, but are greater than those of the wild-type enzyme at EcoRI* sites. Thus, the mutant enzymes use two mechanisms to partially bypass the multiple fail-safe mechanisms that protect against cleavage of genomic DNA in cells carrying the wild-type EcoRI restriction-modification system: (a) binding to EcoRI* sites is more probable than for wild-type enzyme because non-specific DNA is less effective as a competitive inhibitor; (b) the combination of increased affinity and elevated cleavage rate constants at EcoRI* sites makes double-strand cleavage of these sites a more probable outcome than it is for the wild-type enzyme. Semi-quantitative estimates of rates of EcoRI* site cleavage in vivo, predicted using the binding and cleavage constants measured in vitro, are in accord with the observed lethal phenotypes associated with the three mutations.     

Mutational analysis of the SARS virus Nsp15 endoribonuclease: identification of residues affecting hexamer formation

The severe acute respiratory syndrome (SARS) coronavirus virus non-structural protein 15 is a Mn2+-dependent endoribonuclease with specificity for cleavage at uridylate residues. To better understand structural and functional characteristics of Nsp15, 22 mutant versions of Nsp15 were produced in Escherichia coli as His-tagged proteins and purified by metal-affinity and ion-exchange chromatography. Nineteen of the mutants were soluble and were analyzed for enzymatic activity. Six mutants, including four at the putative active site, were significantly reduced in endoribonuclease activity. Two of the inactive mutants had unusual secondary structures compared to the wild-type protein, as measured by circular dichroism spectroscopy. Gel-filtration analysis, velocity sedimentation ultracentrifugation, and native gradient pore electrophoresis all showed that the wild-type protein exists in an equilibrium between hexamers and monomers in solution, with hexamers dominating at micromolar protein concentration, while native gradient pore electrophoresis also revealed the presence of trimers. A mutant in the N terminus of Nsp15 was impaired in hexamer formation and had low endoribonuclease activity, suggesting that oligomerization is required for endoribonuclease activity. This idea was supported by titration experiments showing that enzyme activity was strongly concentration-dependent, indicating that oligomeric Nsp15 is the active form. Three-dimensional reconstruction of negatively stained single particles of Nsp15 viewed by transmission electron microscopic analysis suggested that the six subunits were arranged as a dimer of trimers with a number of cavities or channels that may constitute RNA binding sites.     

Environmental enrichment decreases cell surface expression of the dopamine transporter in rat medial prefrontal cortex

Rats raised in an enriched environmental condition (EC) exhibit a decreased (35%) maximal velocity (V(max)) of [3H]dopamine (DA) uptake in medial prefrontal cortex (mPFC) compared with rats raised in an impoverished condition (IC); however, no differences between EC and IC groups in V(max) for [3H]DA uptake were found in nucleus accumbens and striatum. Using biotinylation and immunoblotting techniques, the present study examined whether the brain region-specific decrease in DA transporter (DAT) function is the result of a reduction in DAT cell surface expression. In mPFC, nucleus accumbens and striatum, total DAT immunoreactivity was not different between EC and IC groups. Whereas no differences in cell surface expression of DAT were found in nucleus accumbens and striatum, DAT immunoreactivity in the biotinylated cell surface fraction of mPFC was decreased (39%) in EC compared with IC rats, consistent with the magnitude of the previously observed decrease in V(max) for [3H]DA uptake in mPFC in EC rats. These results suggest that the decrease in DAT cell surface expression in the mPFC may be responsible for decreased DAT function in the mPFC of EC compared with IC rats, and that there is plasticity in the regulatory mechanisms mediating DAT trafficking and function.     

Identifying the basal angiosperm node in chloroplast genome phylogenies: sampling one's way out of the Felsenstein zone

While there has been strong support for Amborella and Nymphaeales (water lilies) as branching from basal-most nodes in the angiosperm phylogeny, this hypothesis has recently been challenged by phylogenetic analyses of 61 protein-coding genes extracted from the chloroplast genome sequences of Amborella, Nymphaea, and 12 other available land plant chloroplast genomes. These character-rich analyses placed the monocots, represented by three grasses (Poaceae), as sister to all other extant angiosperm lineages. We have extracted protein-coding regions from draft sequences for six additional chloroplast genomes to test whether this surprising result could be an artifact of long-branch attraction due to limited taxon sampling. The added taxa include three monocots (Acorus, Yucca, and Typha), a water lily (Nuphar), a ranunculid (Ranunculus), and a gymnosperm (Ginkgo). Phylogenetic analyses of the expanded DNA and protein data sets together with microstructural characters (indels) provided unambiguous support for Amborella and the Nymphaeales as branching from the basal-most nodes in the angiosperm phylogeny. However, their relative positions proved to be dependent on the method of analysis, with parsimony favoring Amborella as sister to all other angiosperms and maximum likelihood (ML) and neighbor-joining methods favoring an Amborella + Nymphaeales clade as sister. The ML phylogeny supported the later hypothesis, but the likelihood for the former hypothesis was not significantly different. Parametric bootstrap analysis, single-gene phylogenies, estimated divergence dates, and conflicting indel characters all help to illuminate the nature of the conflict in resolution of the most basal nodes in the angiosperm phylogeny. Molecular dating analyses provided median age estimates of 161 MYA for the most recent common ancestor (MRCA) of all extant angiosperms and 145 MYA for the MRCA of monocots, magnoliids, and eudicots. Whereas long sequences reduce variance in branch lengths and molecular dating estimates, the impact of improved taxon sampling on the rooting of the angiosperm phylogeny together with the results of parametric bootstrap analyses demonstrate how long-branch attraction might mislead genome-scale phylogenetic analyses.