The incorporation of glucosamine into enterobacterial core lipopolysaccharide: two enzymatic steps are required

The core lipopolysaccharide (LPS) of Klebsiella pneumoniae is characterized by the presence of disaccharide alphaGlcN-(1,4)-alphaGalA attached by an alpha1,3 linkage to l-glycero-d-manno-heptopyranose II (ld-HeppII). Previously it has been shown that the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS. The presence of GlcNAc instead of GlcN and the lack of UDP-GlcN in bacteria indicate that an additional enzymatic step is required. In this work we identified a new gene (wabN) in the K. pneumoniae core LPS biosynthetic cluster. Chemical and structural analysis of K. pneumoniae non-polar wabN mutants showed truncated core LPS with GlcNAc instead of GlcN. In vitro assays using LPS truncated at the level of d-galacturonic acid (GalA) and cell-free extract containing WabH and WabN together led to the incorporation of GlcN, whereas none of them alone were able to do it. This result suggests that the later enzyme (WabN) catalyzes the deacetylation of the core LPS containing the GlcNAc residue. Thus, the incorporation of the GlcN residue to core LPS in K. pneumoniae requires two distinct enzymatic steps. WabN homologues are found in Serratia marcescens and some Proteus strains that show the same disaccharide alphaGlcN-(1,4)-alphaGalA attached by an alpha1,3 linkage to ld-HeppII.     

Protein transport from the late Golgi to the vacuole in the yeast Saccharomyces cerevisiae

The late Golgi compartment is a major protein sorting station in the cell. Secreted proteins, cell surface proteins, and proteins destined for endosomes or lysosomes must be sorted from one another at this compartment and targeted to their correct destinations. The molecular details of protein trafficking pathways from the late Golgi to the endosomal system are becoming increasingly well understood due in part to information obtained by genetic analysis of yeast. It is now clear that proteins identified in yeast have functional homologues (orthologues) in higher organisms. We will review the molecular mechanisms of protein targeting from the late Golgi to endosomes and to the vacuole (the equivalent of the mammalian lysosome) of the budding yeast Saccharomyces cerevisiae.     

1.15 A crystal structure of the X. tropicalis Spred1 EVH1 domain suggests a fourth distinct peptide-binding mechanism within the EVH1 family

The recently described Spred protein family has been implicated in the modulation of receptor tyrosine kinase signalling. We report the crystal structure of the Enabled/vasodilator-stimulated phosphoprotein homology-1 (EVH1) domain from Xenopus tropicalis Spred1, solved to 1.15 A resolution. This structure confirms that the Spred EVH1 adopts the pleckstrin-homology fold, with a similar secondary structure to Enabled. A translation of one of the peptide-binding groove beta-strands narrows this groove, whilst one end of the groove shows structural flexibility. We propose that Spred1 will bind peptides that are less proline-rich than other EVH1 domains, with conformational changes indicating an induced fit.     

Combining nucleoside analogues to achieve recognition of oligopurine tracts by triplex-forming oligonucleotides at physiological pH

We have used DNase I footprinting to examine DNA triple helix formation at a 12 base pair oligopurine.oligopyrimidine sequence, using oligonucleotides that contain combinations of 2'-aminoethoxy-5-(3-aminoprop-1-ynyl)uridine (bis-amino-U, BAU) and 3-methyl-2-aminopyridine (MeP) in place of T and C, respectively. This combination acts cooperatively to enable high affinity triple helix formation at physiological pH. The affinity depends on the number of substitutions and their arrangement; oligonucleotides in which these analogues are evenly distributed throughout the third strand bind much better than those in which they are clustered together.     

The course of malaria in mice: major histocompatibility complex (MHC) effects, but no general MHC heterozygote advantage in single-strain infections

A general MHC-heterozygote advantage in parasite-infected organisms is often assumed, although there is little experimental evidence for this. We tested the response of MHC-congenic mice (F2 segregants) to malaria and found the course of infection to be significantly influenced by MHC haplotype, parasite strain, and host gender. However, the MHC heterozygotes did worse than expected from the average response of the homozygotes.     

Quantitative trait locus (QTL) isogenic recombinant analysis: a method for high-resolution mapping of QTL within a single population

In the quest for fine mapping quantitative trait loci (QTL) at a subcentimorgan scale, several methods that involve the construction of inbred lines and the generation of large progenies of such inbred lines have been developed (Complex Trait Consortium 2003). Here we present an alternative method that significantly speeds up QTL fine mapping by using one segregating population. As a first step, a rough mapping analysis is performed on a small part of the population. Once the QTL have been mapped to a chromosomal interval by standard procedures, a large population of 1000 plants or more is analyzed with markers flanking the defined QTL to select QTL isogenic recombinants (QIRs). QIRs bear a recombination event in the QTL interval of interest, while other QTL have the same homozygous genotype. Only these QIRs are subsequently phenotyped to fine map the QTL. By focusing at an early stage on the informative individuals in the population only, the efforts in population genotyping and phenotyping are significantly reduced as compared to prior methods. The principles of this approach are demonstrated by fine mapping an erucic acid QTL of rapeseed at a subcentimorgan scale.     

De novo methylation of nucleosomal DNA by the mammalian Dnmt1 and Dnmt3A DNA methyltransferases

In the cell, DNA is wrapped on histone octamers, which reduces its accessibility for DNA interacting enzymes. We investigated de novo methylation of nucleosomal DNA in vitro and show that the Dnmt3a and Dnmt1 DNA methyltransferases efficiently methylate nucleosomal DNA without dissociation of the histone octamer from the DNA. In contrast, the prokaryotic SssI DNA methyltransferase and the catalytic domain of Dnmt3a are strongly inhibited by nucleosomes. We also found that full-length Dnmt1 and Dnmt3a bind to nucleosomes much stronger than their isolated catalytic domains, demonstrating that the N-terminal parts of the MTases are required for the interaction with nucleosomes. Variations of the DNA sequence or the histone tails did not significantly influence the methylation activity of Dnmt3a. The observation that mammalian methyltransferases directly modify nucleosomal DNA provides an insight into the mechanisms by which histone tail and DNA methylation patterns can influence each other because the DNA methylation pattern can be established while histones remain associated to the DNA.     

Molecular characterization of two G protein-coupled receptor splice variants as FLP2 receptors in Caenorhabditis elegans

Two alternatively spliced Caenorhabditis elegans G protein-coupled receptors, T19F4.1a and T19F4.1b, were cloned and functionally characterized. The T19F4.1b receptor protein is 30 amino acids longer than T19F4.1a, and the difference in amino acid constitution is exclusively conferred to the intracellular C-terminal region, suggesting a potential difference in G protein-coupling specificity. Following cloning of the receptor cDNAs into the pcDNA3 vector and stable or transient transfection into Chinese hamster ovary cells, the aequorin bioluminescence/Ca2+ assay was used to investigate receptor activation. This is the first report of the construction of a cell line stably expressing a C. elegans neuropeptide receptor. Our experiments identified both receptors as being cognate receptors for two FMRFamide-related peptides encoded by the flp-2 precursor: SPREPIRFamide (FLP2-A) and LRGEPIRFamide (FLP2-B). Pharmacological profiling using truncated forms of FLP2-A and -B revealed that the active core of both peptides is EPIRFamide. Screening of peptides encoded by other flps did not result in a significant activation of the receptor. In contrast to other C. elegans receptors tested in heterologous expression systems, the functional activation of both T19F4.1a and T19F4.1b was not temperature-dependent. Screening in cells lacking the promiscuous Galpha16 suggests that T19F4.1a and b are both linked to the Gq pathway.     

Perturbation of yeast 3-phosphoglycerate kinase reaction mixtures with ADP: transient kinetics of formation of ATP from bound 1,3-bisphosphoglycerate

3-Phosphoglycerate kinase (PGK) is the first ATP-producing enzyme in glycolysis: ADP + 1,3-bisphosphoglycerate (bPG) <--> ATP + 3-phosphoglycerate (PG). Whereas extensive studies have been carried out on its structure, there is less information about its reaction pathway, which is usually studied in the reverse direction because of the instability of bPG. We studied the transients of the PGK reaction by chemical sampling in a rapid quench flow apparatus, using [gamma-(32)P]ATP, in 30% methanol at 4 degrees C to decrease k(cat). There were two types of experiment, both at low PG concentrations to prevent bPG release. In the first, reaction mixtures were quenched in acid at different times (from 4 ms) and the bPG concentrations were determined. This type gave information about the ATP binding and phospho-transfer steps. In the second, PGK reaction mixtures at equilibrium were perturbed by the injection of ADP, the new mixtures aged for different times and quenched in acid, and the bPG concentrations were determined. This gave information about the kinetics of the binding of ADP to a PGK intermediate. The data from the two types of experiments were fitted to simple schemes and then treated together by a global fitting procedure using a five-step pathway, deduced from previous structural studies. Under our conditions, it appears that (1) a binary PGK.bPG complex is an important intermediate on the reaction pathway, i.e., that ADP is released before bPG, (2) ADP binds to a "closed" conformation in the PGK.bPG complex, and (3) the PGK reaction can be studied in the physiologically important direction without having to handle bPG.