We present a draft sequence of the genome of Aedes aegypti, the primary vector for yellow fever and dengue fever, which at ~1.38 Gbp is ~5-fold larger in size than the genome of the malaria vector, Anopheles gambiae. Nearly 50% of the Aedes aegypti genome consists of transposable elements. These contribute to a ~4-6 fold increase in average gene length and the size of intergenic regions relative to Anopheles gambiae and Drosophila melanogaster. Nevertheless, chromosomal synteny is generally maintained between all three insects although conservation of orthologous gene order is higher (~2-fold) between the mosquito species than between either of them and fruit fly. Three methods have provided transcriptional evidence for 80% of the 15,419 predicted protein coding genes in Aedes aegypti. An increase in genes encoding odorant binding, cytochrome P450 and cuticle domains relative to Anopheles gambiae suggests that members of these protein families underpin some of the biological differences between them.

No abstract

The epsilon subunit of the Escherichia coli replicative DNA polymerase III is the proofreading 3'-5' exonuclease. Structures of its catalytic N-terminal domain (epsilon186) were determined at two pH values (5.8 and 8.5) at resolutions of 1.7-1.8 A, in complex with two Mn(II) ions and a nucleotide product of its reaction, thymidine 5'-monophosphate. The protein structure is built around a core five-stranded beta sheet that is a common feature of members of the DnaQ superfamily. The structures were identical, except for differences in the way TMP and water molecules are coordinated to the binuclear metal center in the active site. These data are used to develop a mechanism for epsilon and to produce a plausible model of the complex of epsilon186 with DNA.

The Escherichia coli replication terminator protein (Tus) binds tightly and specifically to termination sites such as TerB in order to halt DNA replication. To better understand the process of Tus-TerB interaction, an assay based on surface plasmon resonance was developed to allow the determination of the equilibrium dissociation constant of the complex (K D ) and association and dissocation rate constants for the interaction between Tus and various DNA sequences, including TerB, single-stranded DNA, and two nonspecific sequences that had no relationship to TerB. The effects of factors such as the KCl concentration, the orientation and length of the DNA, and the presence of a single-stranded tail on the binding were also examined. The K D measured for the binding of wild type and His 6 -Tus to TerB was 0.5 nM in 250 mM KCl. Four variants of Tus containing single-residue mutations were assayed for binding to TerB and the nonspecific sequences. Three of these substitutions (K89A, R198A, and Q250A) increased K D by 200-300-fold, whereas the A173T substitution increased K D by 4000-fold. Only the R198A substitution had a significant effect on binding to the nonspecific sequences. The kinetic and thermodynamic data suggest a model for Tus binding to TerB which involves an ordered series of events that include structural changes in the protein.

We have physically and genetically characterized 20 symbiotic and 20 auxotrophic mutants of Rhizobium meliloti, the nitrogen-fixing symbiont of alfalfa (Medicago sativa), isolated by transposon Tn5 mutagenesis. A "suicide plasmid" mutagenesis procedure was used to generate TN-5-induced mutants, and both auxotrophic and symbiotic mutants were found at a frequency of 0.3% among strains containing random TN5 insertions. Two classes of symbiotic mutants were isolated: 4 of the 20 formed no nodules at all (Nod-), and 16 formed nodules which failed to fix nitrogen (Fix-). We used a combination of physical and genetic criteria to determine that in most cases the auxotrophic and symbiotic phenotypes could be correlated with the insertion of a single Tn5 elements. Once the Tn5 element was inserted into the R. meliloti genome, the frequency of its transposition to a new site was approximately 10-8 and the frequency of precise excision was less than 10-9. In approximately 25% of the mutant strains, phage Mu DNA sequences, which originated from the suicide plasmid used to generate the Tn5 transpositions, were also found in the R. meliloti genome contiguous with Tn5. These later strains exhibited anomalous conjugation properties, and therefore we could not correlate the symbiotic phenotype with a Tn5 insertion. In general, we found that both physical and genetic tests were required to fully characterize transposon-induced mutations.

The mechanism of dissociation reactions induced by calcium chelators has been studied for complexes of Drosophila calmodulin with target peptides, including four derived from the skeletal muscle myosin light chain kinase target sequence. Reactions were monitored by fluorescence stopped-flow techniques using a variety of intrinsic probes and the indicator Quin2. For most of the complexes, apparently biphasic kinetics were observed in several fluorescence parameters. The absence of any obvious relationship between dissociation rates and peptide affinities implies kinetic control of the dissociation pathway. A general mechanism for calcium and peptide dissociation was formulated and used in numerical simulation of the experimental data. Unexpectedly, the rate of the slowest step decreases with increasing [peptide]/[calmodulin] ratio. Numerical simulation shows this step could contain a substantial contribution from a reversible relaxation process (involving the species Ca 2 -calmodulin-peptide), convolved with the following step (loss of C-terminal calcium ions).The results indicate the potentially key kinetic role of the partially calcium-saturated intermediate species.They show that subtle changes in the peptide sequence can have significant effects on both the dissociation rates and also the dissociation pathway. Both effects could contribute to the variety of regulatory behavior shown by calmodulin with different target enzymes.

The core of DNA polymerase III, the replicative polymerase in Escherichia coli, consists of three subunits (alpha, epsilon, and theta). The epsilon subunit is the 3'-5' proofreading exonuclease that associates with the polymerase (alpha) through its C-terminal region and theta through a 185-residue N-terminal domain (epsilon 186). A spectrophotometric assay for measurement of epsilon activity is described. Proteins epsilon and epsilon 186 and the epsilon 186.theta complex catalyzed the hydrolysis of the 5'-p-nitrophenyl ester of TMP (pNP-TMP) with similar values of k(cat) and K(M), confirming that the N-terminal domain of epsilon bears the exonuclease active site, and showing that association with theta has little direct effect on the chemistry occurring at the active site of epsilon. On the other hand, formation of the complex with theta stabilized epsilon 186 by approximately 14 degrees C against thermal inactivation. For epsilon 186, k(cat) = 293 min(-)(1) and K(M) = 1.08 mM at pH 8.00 and 25 degrees C, with a Mn(2+) concentration of 1 mM. Hydrolysis of pNP-TMP by epsilon 186 depended absolutely on divalent metal ions, and was inhibited by the product TMP. Dependencies on Mn(2+) and Mg(2+) concentrations were examined, giving a K(Mn) of 0.31 mM and a k(cat) of 334 min(-1) for Mn(2+) and a K(Mg) of 6.9 mM and a k(cat) of 19.9 min(-1) for Mg(2+). Inhibition by TMP was formally competitive [K(i) = 4.3 microM (with a Mn(2+) concentration of 1 mM)]. The pH dependence of pNP-TMP hydrolysis by epsilon 186, in the pH range of 6.5-9.0, was found to be simple. K(M) was essentially invariant between pH 6.5 and 8.5, while k(cat) depended on titration of a single group with a pK(a) of 7.7, approaching limiting values of 50 min(-1) at pH <6.5 and 400 min(-1) at pH >9.0. These data are used in conjunction with crystal structures of the complex of epsilon 186 with TMP and two Mn(II) ions bound at the active site to develop insights into the mechanisms of pNP-TMP hydrolysis by epsilon at high and low pH values.

The sliding clamp of the Escherichia coli replisome is now understood to interact with many proteins involved in DNA synthesis and repair. A universal interaction motif is proposed to be one mechanism by which those proteins bind the E. coli sliding clamp, a homodimer of the beta subunit, at a single site on the dimer. The numerous beta(2)-binding proteins have various versions of the consensus interaction motif, including a related hexameric sequence. To determine if the variants of the motif could contribute to the competition of the beta-binding proteins for the beta(2) site, synthetic peptides derived from the putative beta(2)-binding motifs were assessed for their abilities to inhibit protein-beta(2) interactions, to bind directly to beta(2), and to inhibit DNA synthesis in vitro. A hierarchy emerged, which was consistent with sequence similarity to the pentameric consensus motif, QL(S/D)LF, and peptides containing proposed hexameric motifs were shown to have activities comparable to those containing the consensus sequence. The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to beta(2) in various stages or circumstances of DNA replication and repair.