Rapid analysis of large protein-protein complexes using NMR-derived orientational constraints: the 95 kDa complex of LpxA with acyl carrier protein

Characterization of protein-protein interactions that are critical to the specific function of many biological systems has become a primary goal of structural biology research. Analysis of these interactions by structural techniques is, however, challenging due to inherent limitations of the techniques and because many of the interactions are transient, and suitable complexes are difficult to isolate. In particular, structural studies of large protein complexes by traditional solution NMR methods are difficult due to a priori requirement of extensive assignments and a large number of intermolecular restraints for the complex. An approach overcoming some of these challenges by utilizing orientational restraints from residual dipolar couplings collected on solution NMR samples is presented. The approach exploits existing structures of individual components, including the symmetry properties of some of these structures, to assemble rapidly models for relatively large protein-protein complexes. An application is illustrated with a 95 kDa homotrimeric complex of the acyltransferase protein, LpxA (UDP-N-acetylglucosamine acyltransferase), and acyl carrier protein. LpxA catalyzes the first step in the biosynthesis of the lipid A component of lipopolysaccharide in Gram-negative bacteria. The structural model generated for this complex can be useful in the design of new anti-bacterial agents that inhibit the biosynthesis of lipid A.     

Genic mutation rates in mammals: local similarity,chromosomal heterogeneity,and X-versus-autosome disparity

The reduction of mutation rates on the mammalian X chromosome relative to autosomes is most often explained in the literature as evidence of male-driven evolution. This hypothesis attributes lowered mutation rates on the X chromosome to the fact that this chromosome spends less time in the germline of males than in the germline of females. In contrast to this majority view, two articles argued that the patterns of mutation rates across chromosomes are inconsistent with male-driven evolution. One article reported a 40% reduction in synonymous substitution rates (Ks) for X-linked genes relative to autosomes in the mouse-rat lineage. The authors argued that this reduction is too dramatic to be explained by male-driven evolution and concluded that selection has systematically reduced mutation rate on the X chromosome to a level optimal for this male-hemizygous chromosome. More recently, a second article found that chromosomal mutation rates in both the human-mouse and mouse-rat lineages were so heterogeneous that the X chromosome was not an outlier. Here again, the authors argued that this is at odds with male-driven evolution and suggested that selection has modulated chromosomal mutation rates to locally optimal levels, thus extending the argument of the first mentioned article to include autosomes. Here, we reexamine these conclusions using mouse-rat and human-mouse coding-region data. We find a more modest reduction of Ks on the X chromosome, but our results contradict the finding that the X chromosome is not distinct from autosomes. Multiple statistical tests show that Ks rates on the X chromosome differ systematically from the autosomes in both lineages. We conclude that the moderate reduction of mutation rate on the X chromosome of both lineages is consistent with male-driven evolution; however, the large variance in mutation rates across chromosomes suggests that mutation rates are affected by additional factors besides male-driven evolution. Investigation of mutation rates by synteny reveals that synteny blocks, rather than entire chromosomes, might represent the unit of mutation rate variation.     

Molecular evolution of P transposable elements in the genus Drosophila. III. The melanogaster species group

Phylogenetic relationships were determined for 76 partial P-element sequences from 14 species of the melanogaster species group within the Drosophila subgenus Sophophora. These results are examined in the context of the phylogeny of the species from which the sequences were isolated. Sequences from the P-element family fall into distinct subfamilies, or clades, which are often characteristic for particular species subgroups. When examined locally among closely related species, the evolution of P elements is characterized by vertical transmission, whereby the P-element phylogeny traces the species phylogeny. On a broader scale, however, the P-element phylogeny is not congruent with the species phylogeny. One feature of P-element evolution in the melanogaster group is the presence of more than one P-element subfamily, differing by as much as 36%, in the genomes of some species. Thus, P elements from several individual species are not monophyletic, and a likely explanation for the incongruence between P-element and species phylogenies is provided by the comparison of paralogous sequences. In certain instances, horizontal transfer seems to be a valid alternative explanation for lack of congruence between species and P-element phylogenies. The canonical P-element subfamily, which represents the active, autonomous transposable element, is restricted to D. melanogaster. Thus, its origin clearly lies outside of the melanogaster species group, consistent with the earlier conclusion of recent horizontal transfer.      

Stabilization of pet operon plasmids and ethanol production in Escherichia coli strains lacking lactate dehydrogenase and pyruvate formate-lyase activities.

In the last decade, a major goal of research in biofuels has been to metabolically engineer microorganisms to ferment multiple sugars from biomass or agricultural wastes to fuel ethanol. Escherichia coli strains genetically engineered to contain the pet operon (Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase B genes) produce high levels of ethanol. Strains carrying the pet operon in plasmid (e.g., E. coli B/pLOI297) or in chromosomal (e.g., E. coli KO11) sites require antibiotics in the media to maintain genetic stability and high ethanol productivity. To overcome this requirement, we used the conditionally lethal E. coli strain FMJ39, which carries mutations for lactate dehydrogenase and pyruvate formate lyase and grows aerobically but is incapable of anaerobic growth unless these mutations are complemented. E. coli FBR1 and FBR2 were created by transforming E. coli FMJ39 with the pet operon plasmids pLOI295 and pLOI297, respectively. Both strains were capable of anaerobic growth and displayed no apparent pet plasmid losses after 60 generations in serially transferred (nine times) anaerobic batch cultures. In contrast, similar aerobic cultures rapidly lost plasmids. In high-cell-density batch fermentations, 3.8% (wt/vol) ethanol (strain FBR1) and 4.4% (wt/vol) ethanol (strain FBR2) were made from 10% glucose. Anaerobic, glucose-limited continuous cultures of strain FBR2 grown for 20 days (51 generations; 23 with tetracycline and then 28 after tetracycline removal) showed no loss of antibiotic resistance. Anaerobic, serially transferred batch cultures and high-density fermentations were inoculated with cells taken at 57 generations from the previous continuous culture. Both cultures continued to produce high levels of ethanol in the absence of tetracycline. The genetic stability conferred by selective pressure for pet-containing cells without requirement for antibiotics suggests potential commercial suitability for E. coli FBR1 and FBR2.     

Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.